IGH@ proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between IGH@ proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression. IGH@ proto-oncogene translocation is a known genomic driver in several blood cancers. Here, the authors show that IGH-DUX4 translocation occurs on the silenced IGH allele avoiding toxic high-level expression of DUX4 in B-ALL.The  IGH (immunoglobulin heavy chain) follows a one B-cell, one antibody paradigm by first epigenetically silencing both alleles, then activating one allele during VDJ recombination so that functional IGH is expressed from only one active IGH allele in pre-B (precursor B) cells4. In lymphoid lineage cancer like B-cell acute lymphoblastic leukemia (B-ALL)5, lymphoma6, and multiple myeloma7, IGH@ proto-oncogene translocation is a common driver event. There is, however, limited knowledge about the interplay between IGH@ oncogenic translocation and allelic exclusion of IGH  partly due to the technical challenges in delineating allelic specificity at the highly complex IGH locus (standard next-generation sequencing [NGS] technology can only generate short-read sequence data ranging from 100 to 150 bp).In B-cell development, the allelic exclusion of IGH-DUX4 translocation is one such oncogenic event, defining a B-ALL subtype with a distinct expression profile10. DUX4 is a transcription factor located within the GC-rich D4Z4 repeat array at the subtelomeric regions of 4q35 and 10q26, which are characterized by high levels of repression12. It is expressed only during events associated with major chromatin relaxation, i.e., in early embryos at cleavage stage (2/4/8-cell embryos)13 or after loss of repression of the D4Z4 macrosatellite repeat in myoblasts of individuals with facioscapulohumeral muscular dystrophy (FSHD)15. The IGH-DUX4 subtype accounts for ~7% of pediatric B-ALLs in which an inter-chromosomal translocation repositions DUX4 within the vicinity of the IGH enhancer (Eμ), the native enhancer of IGH gene (Igμ) in pre-B cell16. IGH-DUX4 translocation is a clonal event acquired early in leukemogenesis of this B-ALL subtype, resulting in an aberrant activation of DUX4 which is absent from other B-ALL subtypes9.IGH@ oncogenic translocation and allelic exclusion of IGH in Nalm6, a B-ALL cell line that harbors IGH-DUX4 translocation. We use long-read technology which overcomes the limitation of short-read sequencing to evaluate the allele specificity of gene expression. Epigenetic states and enhancer–promoter interactions are analyzed using data generated from ChIP-seq, ATAC-seq, and 3-D genome assays. Significant allelic imbalance is detected on the wild type over the IGH-DUX4 haplotype, showing that IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro assays suggest this may reduce the oncogenic stress of high-level expression of DUX4. Patient samples of IGH-DUX4 B-ALL have similar expression profiles and IGH breakpoints as Nalm6, suggesting this could be a common mechanism; further analyses of haplotype structure and epigenetic profiling are required.In this study, we investigate the interplay between IGH-DUX4 translocation and human embryos17, we found that DUX4 expression is much higher compared with normal expression in human cleavage stage embryos . Consistent with our observation in IGH-DUX4 B-ALL patient samples, DUX4 expression in Nalm6 is much higher compared with its normal expression in human cleavage stage embryos  Iso-Seq, a method that generates full-length transcripts; (2) 10X Chromium whole-genome sequencing (WGS), which incorporates barcodes into large DNA fragments prior to WGS thereby producing the so-called “digital” long WGS reads; and (3) stranded total RNA-seq. We also performed epigenetic and chromosome conformation capture assays to evaluate the allele specificity of epigenetic states and enhancer–promoter interactions. These include (1) H3K27ac ChIP-seq, ATAC-seq, and whole-genome bisulfite sequencing (WGBS); (2) genome-wide 3-D genome assays including Hi-C and H3K27ac HiChIP; and (3) deep sequencing of targeted chromosome conformation capture (Capture-C).To resolve the mapping ambiguity caused by high repeat content in DUX4 copies were translocated between IGHJ4 and IGHD2–15 in Nalm6, resulting in expression of a DUX4 protein with its last 16 amino acids replaced by IGH8. Incorporating this knowledge in our analysis, we first assembled the haplotype of IGH-DUX4 translocation by selecting reads near the rearranged region using Chromium molecular barcodes. This resulted in a ~23 kb DNA sequence comprised of IGHM, the IGH intronic enhancer (Eμ), two copies of the translocated DUX4 sequence, and flanking sequences near the two translocation breakpoints  sequence identity. RNA-seq reads share 100% identity to the assembled DUX4 contig, supporting exclusive transcription of DUX4L13 activated by translocation from chromosome 10 to IGH in Nalm6 , which is also contained in the functional Igμ transcript. Bidirectional transcription of sense and antisense DUX4 was also confirmed in our stranded RNA-seq data . Haplotype phasing from 10X Chromium WGS data demonstrate two haplotypes harboring alleles “T-G-G” and “A-A-A” of these three SNPs. The full-length transcripts generated by Iso-Seq showed that functional Igμ was transcribed exclusively from the “A-A-A” haplotype  while the antisense DUX4 was transcribed from the “T-G-G” haplotype that harbors the IGH-DUX4 translocation  compared with the IGH-DUX4 haplotype : expression of the wild-type allele is 7.4 and 10.4-fold of that of the IGH-DUX4 haplotype allele at the two exonic SNPs rs1059713 and rs1136534, respectively. A similar pattern was found in H3K27ac and ATAC-seq reads which measure enhancer activity and chromatin accessibility, respectively 16. We confirmed the interactions of Eμ-Igμ promoter and Eμ-DUX4 by performing genome-wide Hi-C  is in fact much longer than that (~4 kb) between Eμ and DUX4 on the IGH-DUX4 haplotype . To further evaluate the effects of DUX4 overexpression in fully transformed leukemia cells, we introduced DUX4 into Nalm6 cells which already harbor the IGH-DUX4 translocation, confirming GFP-tagged DUX4 protein expression by western blot . We therefore conclude that while low levels of the DUX4 protein are tolerable, high levels induce apoptosis providing a biologic rationale for expression from the repressive haplotype.Our analysis on the transcriptome, epigenome and 3D genome shows that in Nalm6 the ene Fig. . Apoptoslot Fig. . The resIGH-DUX4 translocation during B-cell development  cells where both IGH alleles undergo D-to-J rearrangement. Structural variation (SV) breakpoints of IGH-DUX4 in Nalm6 occurred in the IGH D-J junction, suggesting that the IGH translocation arise at the pro-B cell stage as demonstrated previously by SV breakpoint analysis in IGH rearranged multiple myeloma patient samples23. Consistent with this, prior studies by our group and the others showed that the IGH-DUX4 fusions are clonal events acquired early in leukemogenesis9. At this stage, both IGH alleles remain methylated. In late pro-B and pre-B cell stage, one of the IGH alleles is randomly selected for activation by demethylation and hyperacetylation followed by the VDJ rearrangement. If an IGH-DUX4 rearranged allele were selected for activation—which is possible as not all B-ALLs express functional IGH25, the resulting highly expressed DUX4 would likely be too toxic to permit the survival of leukemia cells, leading to cell death  with a positive correlation  between Igu and DUX4 expression  is significantly lower than that of Igμ—both patterns match what we observed in Nalm6. The consistency between patient data and the Nalm6 cell line suggest that IGH@ proto-oncogene translocation on the silenced allele, discovered in Nalm6 through comprehensive analysis on haplotype structure and epigenetic profiling, could also occur in B-ALL patient samples. Further studies employing haplotype analysis and epigenomic profiling using patient samples or patient-derived xenograft mouse models are needed to provide additional support for this hypothesis.Our analysis of RNA-seq data generated from 54 B-ALL pediatric patient samples with UX4 Fig.  and CRLF13 were downloaded from the GEO database by SRA toolkit v2.8.2-1. The data were aligned to hg19 (GRCh37-lite) using STAR v2.529. Published RNA-seq data of 32 IGH-DUX4 and 24 IGH-CRLF2 B-ALL patients were used for the analysis28, which were available in EGA  and dbGaP (phs000218). FPKM was calculated for each gene based on the gene models in GENCODE v19 using HTseq-count30. Since there are many copies of DUX4 in the human genome, in order to evaluate DUX4 gene expression level, all the reads were mapped to DNA segment of a single DUX4 locus , then the mapped read counts were used to calculate FPKM. Our local assembly of IGH-DUX4 haplotype derived from the Chromium WGS showed DUX4L13 on the reference genome is the best match to the translocated DUX4. For Igμ, different B cells use different V/D/J components; however, the constant region of Igμ (IGHM) is used in all pre-B cells1. The FPKM of IGHM was therefore used to evaluate Igμ gene expression.RNA-seq data (GSE85632) for human cleavage cellsHigh molecular weight genomic DNA of Nalm6  was extracted using Qiagen MagAttract HMW DNA Kit. 1.0 ng HMW gDNA was input, and each molecule was captured by a gel bead which contained specific barcode to distinguish different molecules on 10X Genomics Chromium Controller, then amplified by isothermal incubation. The barcoded DNA was purified, then end repaired, dA-tailed and adaptor ligated using chromium genome library kit. Finally, the library was purified and enriched by index PCR amplification then sequenced paired-end 151 cycles on Illumina HiSeq4000.https://support.10xgenomics.com/genome-exome/software/pipelines/latest/using/wgs) was used to perform variant calling and phasing. First, we mapped the reads to hg19 with Lariat using default parameters. Second, Freebayes v0.9.21 was used to call haplotype-based variants. Finally, we filtered the variants using bcftools v1.1 (-e ‘(%QUAL<= 15 || (AF[0]>0.5 && %QUAL<50))’ and -e ‘(AO[0]<2 || AO[0]/(AO[0]+RO) <0.15)’).The Long Ranger 2.0.0 pipeline  were selected. Second, we obtained the barcodes from the selected reads, only keeping barcodes with ≥ 20 reads. Third, all the reads with these barcodes in the whole library were selected, even where the read was unmapped or was mapped to non-IGH regions. Fourth, de novo DNA assembly was performed based on the selected reads using Supernova v1.1.432. We used GRCh38 for this analysis because the annotation on IGH and DUX4 is much improved on this more recent version of the reference genome. An 8063 bp contig was mapped to the IGHM region  was used in this linking region . In summary, we manually assembled a 23,361 bp DNA segment, including IGHM, the IGH intronic enhancer (Eμ), the translocated DUX4 sequence, and the sequence near the breakpoints.The Chromium molecular barcodes were used to select reads near the DUX4 region was best matched to DUX4L13. DUX4L13 showed exactly the same as our assembled sequence. The other DUX4 copies in hg19 showed at least one mismatch with the assembled sequence.We mapped the ~23 kb sequence to hg19 and found the assembled One microgram of total RNA per reaction was reverse transcribed using the Clontech SMARTer cDNA synthesis kit. Four reverse transcription (RT) reactions were processed in parallel. PCR optimization was used to determine the optimal amplification cycle number for the downstream large-scale PCR reactions. A single primer (primer IIA from the Clontech SMARTer kit 5′-AAGCAGTGGTATCAACGCAGAGTAC-3′) was used for all PCR reactions post-RT.https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Iso-Seq%E2%84%A2-Template-Preparation-for-Sequel-Systems.pdf).Large-scale PCR products were purified with either 1× or 0.4× AMPure PB beads (1× or 0.4× cDNA library hereafter) and the bioanalyzer was used for QC. Equimolar ratios of the 1× and 0.4× cDNA libraries were pooled to generate the non-size-selected SMRTBell library. A total of 3.5 µg of 1× cDNA library was input into size fractionation using the Sage BluePippin system. A 4.5–10 kb size fraction was eluted, re-amplified and cleaned with AMPure PB beads. A SMRTBell library was constructed using 2.3 µg of size-selected cDNA  were sequenced on the PacBio Sequel platform using 2.0 chemistry.29 with recommended parameters for Iso-Seq . The majority of the mapped reads contained IGHM exons , and these reads were in-frame coding for functional Igμ. Two IGH-DUX4 fusion transcripts were found, and the shorter sequence was supported by 3′ RACE experiment by Yasuda et al.8. Two antisense DUX4 transcripts containing IGHM exons were also identified  reads  were generated. Then all the reads were mapped to the assembled DNA segment using STAR v2.59 and available in EGA (EGAS00001000654).Total stranded RNA-seq in Nalm6 were from our previously published work33. Briefly, around 50,000 Nalm6 cells were used for nuclei preparation. Chromatin fragmentation and library construction were carried out using the Nextera DNA sample preparation kit . DNA library sequencing was done on an Illumina Hiseq 2500 with 75 bp paired-end reads. The reads were trimming for Nextera adapter by cutadapt  and aligned to human genome hg19 (GRCh37-lite) by BWA 34, duplicated reads were then marked with Picard (version 2.6.0-SNAPSHOT)35 and only non-duplicated proper paired reads have been kept by samtools (parameter “-q 1 -F 1804” version 1.2)36. After adjustment of Tn5 shift (reads were offset were offset by + 4 bp for the sense strand and −5 bp for the antisense strand) we separated reads into nucleosome free, mononucleosome, dinucleosome, tri-nucleosome as described in Buenrostro et al.37 by fragment size and generated bigwig files by using the center 80 bp of fragments and scale to 30 M nucleosome free reads. We observed reasonable nucleosome free peaks and pattern of mono-, di-, tri-nucleosome on IGV (version 2.4.13)38. The samples have more than 20 M nucleosome free reads so we conclude the data qualities are good and have enough depth to not miss any chromatin accessible regions.For ATAC sequencing, we followed the protocol established by Buenrostro et al.34, duplicated reads were then marked with Picard (version 2.6.0-SNAPSHOT)35 and only non-duplicated reads have been kept by samtools (parameter “-q 1 -F 1024” version 1.2)36. We followed ENCODE guideline39 for quality control. Briefly, we used SPP40 to draw cross-correlation plot and calculated relative strand correlation value (RSC). We then estimate the fragment size from cross-correlation plot and extended each read to the estimated fragment size to generate bigwig files . Observed clear peak shape along with RSC > 1 and ~40 M (ENCODE criterion 10 M) unique mapped reads, we conclude our IP sample for H3K27ac is good.For the H3K27ac ChIP-seq, a frozen cell pellet containing 10 million cells was sent to Active Motif for ChIP and library preparation. The sample was divided into an aliquot for ChIP using an antibody to H3K27ac (Active Motif) and an input control. Single-end sequencing was performed using an Illumina NextSeq 500 generating 76 cycles for each sequencing read. The reads were mapped to human genome hg19 (GRCh37-lite) by BWA In total, 200 ng genomic DNA of Nalm6 was sheared around 400 bp using Covaris LE220 and followed by bisulfite conversion using EZ DNA Methylation-Gold Kit. The fragmented, bisulfite-converted single-strand DNA was tailed and ligated of truncated adapter one to 3′ end, primer extended and truncated of adapter two at the bottom strand only. Finally, the library was purified and enriched by six cycles of index PCR amplification then sequenced paired-end 151 cycles on Illumina NovaSeq 6000.41 (-q 20 -u -v 0.08 -w 100 -z 33). The mapping rate was 92.9%, and the bisulfite conversion rate was 99.3% in mitochondria. For allele-specific DNA methylation analysis, in IGHM region, only the heterozygous SNPs rs1136534 was in CG island. All 9 CG positions near the SNP (left and right 45 bp region) were considered. Because the SNP rs1136534 located in CG content, only the reads with meaningful orientation overlapped with the SNP were considered. For each selected read, we first identified if the read was a wild type or IGH-DUX4 rearranged allele before assessing the number of methylated or unmethylated reads to define coverage and the methylation ratio at the 9 CG positions.The reads were mapped to hg19 using bsmap v2.74The Nalm6 cell line was cultured under recommended conditions to about 80% confluence. Five million cells were crosslinked with 1% formaldehyde for 10 min at room temperature, then digested with 125 units of MboI, and labeled by biotinylated nucleotides and proximity-ligated. After reverse crosslinking, ligated DNA was purified and sheared to 300–500 bp, then ligation junctions were pulled down with streptavidin beads and prepared as general Illumina library. The Hi-C sample was sequenced paired-end 76 cycles on Illumina Hiseq 4000.42 with default parameters. Of ~2.5 billion Hi-C reads, 60.9% were Hi-C contact reads. Because many copies of DUX4 exist in the reference genome, in order to show the reasonable signals in the DUX4 region, no minimum mapping quality was required. The data matrices were further extracted from .hic files with the dump function from juicer tools, and the data were visualized with the Hi-C viewer under development though the Pediatric Cancer (PeCan) data portal43.The reads were mapped to hg19 and processed using Juicer v1.5IGH intronic enhancer (Eμ) lies between the tandem repeat region and IGHJ region44. In order to extract the contact reads between Eμ and Igμ promoter from Hi-C data, we defined Eμ and Igμ promoter as the regions with highly enriched H3K27ac/ATAC-seq reads, marked as red boxes in Supplementary Fig. DUX4 region, to quantify Eμ-DUX4 interaction reads, all the read pairs that one read falls in Eμ and the other in any DUX4 array region annotated by Gencode V28lift37 were defined as the read pairs supported Eμ-DUX4 interaction.The The Nalm6 cell line was cultured under recommended conditions to about 80% confluence. DNA from 10 million cells were crosslinked with 1% formaldehyde for 10 min at room temperature, then digested with 375 units of MboI, labeled by biotinylated nucleotides and proximity-ligated. After being sheared by Covaris, chromatin immunoprecipitated (ChIP) with H2K27ac antibody, the ChIP DNA was de-crosslinked and purified by Zymo Genomic GNA Clean & Concentrator kit. Finally, after streptavidin beads pull-down and Illumina library construction by Kapa Hyper Prep Kit, hiChIP library was sequenced paired-end 76 cycles on Illumina NextSeq 500.45 was used for H3K27ac HiChIP data analysis. Of ~371.6 million reads, 75.6% were contact read. The same defined enhancer/promoter region and the same strategy for Hi-C data analysis were used to quantify Eμ-DUX4 interaction reads and Eμ-Igμ promoter interaction reads in H3K27ac HiChIP data.HiC-ProCapture-C was designed to identify the regions interacting with Eμ. There were three DpnII cutting sites   Fig. . This reDUX4 region and Igμ promoter region were used to quantify Eμ-DUX4 and the Eμ-Igμ interaction. Although many DUX4 copies exist in human genome, only one peak of 56 reads at DUX4L13  was found  using BWA34, resulting in an increase of DUX4L13 peak coverage to 75. Therefore, the fold change of interaction intensity between the Eμ-Igμ promoter and Eμ-DUX4 was estimated to be 13 fold (953 vs. 75).The highest peak at IGH-DUX4, the linear distance between Eμ and DUX4 is ~4 kb  was shown in Supplementary Fig. DUX4 copies were inserted into IGH D and J region” was shown as blue box. Most IGHV regions were deleted on the wild-type IGH allele based on well-known allelic exclusion of IGH3. From the coverage of Chromium WGS, the big IGHV region “C” (marked as red bar) has one allele deletion, suggesting sequence at region “C” is on the non-wild-type IGH allele. The region “C” does not interact with IGHM/enhancer region “A” (marked as green bar) but interact with chr10 subtelomere (marked as cyan bar) shown as yellow box, suggesting a reciprocal translocation between chr10 subtelomere and IGH region. A model of the two translocation events between chr10 subtelomere and the non-wild-type  IGH allele was shown in Supplementary Fig. A large-scale Hi-C plot between chr10 subtelomere (1.4 Mb) and chr14 DUX4 and ACTB cDNA were cloned into the MSCV-IRES-eGFP retroviral vector. Ecotropic envelope-pseudotyped virus was generated using the Phoenix™-Eco cell line  as per manufacturers instructions. Murine bone marrow from 4- to 6-week-old C57BL/6 mice was harvested, lineage depleted as per manufacturers instructions , and cultured in the presence of murine IL-3 (10 ng/ml), IL-6 (30 ng/ml), and SCF (50 ng/ml)  for 24 h prior to transduction on RetroNectin (Takara Bio Inc.)49. Thirty-six to forty-eight hours following transduction with a single hit of virus, cells were harvested and analyzed by flow cytometry. Briefly, cells were washed and stained with annexin V conjugated to APC  for 15 min at 4 °C, washed and resuspended in 4′,6-diamidino-2-phenylindole containing solution (1 μg/ml DAPI) for analysis using FACS LSR II D . All animal studies were maintained and treated in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at St. Jude Children’s research hospital. For the Nalm6 cell line, a FLAG-tagged full-length DUX4 gene was cloned into the lentiviral construct pCDH . VSV-G pseudotyped virus was generated by transient transfection as per manufacturer’s instructions . Thirty-six hours following transduction with a single hit of virus, cells were harvested and analyzed by flow cytometry and described above. GFP-positive populations were flow sorted for western blot analysis to confirm expression of DUX4 from the lentiviral construct. The details of gating strategy for mice bone marrow and Nalm6 can be found in Supplementary Fig. For the murine experiments, full-length ine IL-3  ng/ml, IIGH-DUX4 B-ALL patients, the C-terminal truncation of DUX4 caused by translocation varies significant, from 4aa to ~200aa  based on previous studies by us and others10 (https://pecan.stjude.cloud/proteinpaint/DUX4). In a recent study, Dong et al. found that the wild-type DUX4 and IGH-DUX4 share similar DNA binding mechanism10. The crystal structure of Apo-DUX4HD2 and DNA-bound complex suggests that the N-terminal double homeobox domains are essential for IGH-DUX4 driven transactivation and B-ALL leukemogenesis. Combined, these data suggest the enhancer hijacking leading to expression of the DUX4 peptide is the real oncogenic effect. Therefore, the wild-type DUX4 was utilized in our apoptosis experiments.In Total protein was extracted from GFP positive flow sorted Nalm6 cells with RIPA lysis buffer and blotted with anti-FLAG  and anti-Beta Actin . Anti-mouse IgG superclonal-HRP  and anti-rabbit-HRP  were used as secondary antibodies for FLAG and Beta Actin antibodies, respectively. Uncropped and unprocessed scans can be found in Supplementary Fig. Further information on research design is available in the Supplementary InformationTransparent Peer Review FileReporting Summary


Streptococcus pyogenes CRISPR/Cas system has found widespread applications as a gene‐editing and regulatory tool for the simultaneous delivery of the Cas9 protein and guide RNAs into the cell, thus making the recognition of specific DNA sequences possible. The recent study that shows that Cas9 can also bind to and cleave RNA in an RNA‐programmable manner is suggestive of potential utility of this system as a universal nucleic‐acid recognition tool. To increase the signal intensity of the CRISPR/Cas system, a signal amplification technique has to be exploited appropriately; this requirement is also a challenge for the detection of DNA or RNA. Furthermore, the CRISPR/Cas system may be used to detect point mutations or single‐nucleotide variants because of the specificity of the recognition between the target sequence and the CRISPR/Cas system. These lines of evidence make this technique capable of detecting pathogens during infection via analysis of their DNA or RNA. Thus, here we summarize applications of the CRISPR/Cas system to the recognition and detection of DNA and RNA molecules as well as the signal amplification. We also describe its potential ability to detect mutations and single‐nucleotide variants. Finally, we sum up its applications to testing for pathogens and potential barriers for its implementation.The  The researchers found it possible to combine these two RNAs into a chimeric single guide RNA (known as sgRNA) that can effectively direct Cas9 to specific DNA targets. The rules used by Cas9 to search for a DNA target are not complicated, the specificity is determined only by a 20‐nucleotide (nt) sequence in the sgRNA that hybridizes with the target DNA in the presence of a DNA protospacer‐adjacent motif (PAM) in the complementary region close to the target site.Clustered regions of interspersed palindromic repeats with Cas9 (CRISPR/Cas9) is an antiviral defense system first found in 2MUC4 gene and concluded that 26 to 36 sgRNAs are sufficient to detect a non‐repetitive genomic locus by means of CRISPR. They also labelled different regions of the same gene or different genes with multiple sgRNAs and reported that the spots of imaging via two genes increased whereas those of the same gene did not, suggesting that CRISPR can label multiple genomic elements at the same time. On the basis of this observation, Wulan DengS. pyogenes Cas9 whose PAM is required only for the “NGG” sequence, many other bacterial Cas9 proteins recognize more complicated PAM sequences, which makes the target sequence more demanding. Consequently, to circumvent these issues, Yi Fu et alDNA fluorescence in situ hybridization (FISH) has been widely used to visualize sequence‐specific genes for research and diagnostic purposes. Nonetheless, it requires heat treatments and formamide to denature double‐stranded DNA (dsDNA) to enable probe hybridization; these treatments may affect integrity of the biological structure and organization of the genome. Accordingly, Baohui ChenIn view of the development of DNA detection technologies, from repetitive to non‐repetitive sequences, the challenge was to enhance the signal released from the locus of the target sequence labelled with re‐engineered sgRNA/dCas9–viral RNA‐binding protein–fluorescent protein complex. Comparison of the detection of repetitive and non‐repetitive sequences revealed that the former yields an obvious signal amplification effect because its abundant copies can combine with much more sgRNA molecules, which should produce a stronger signal. Nonetheless, the latter was not able to produce such a strong signal this way. Therefore, the researchers devised two other methods to amplify the signal: one involves transduction of large amounts of sgRNAs targeting the same gene in different regions, and the other involves attaching more fluorescent proteins to one single sgRNA. The limitations of the first method are the following: (a) with the increase in the number of sgRNAs, the off‐target effects will become more serious and ineluctable; and (b) it is technically difficult to deliver so many sgRNAs into a cell. It seemed that the second method is suitable for detection of a single non‐repetitive sequence but still required four sgRNAs, and lattice light sheet microscopy was needed to obtain a reliable signal. From the single loci to simultaneous multilocus detection, various improvements have been made to add various fluorescent colours though the method was still restricted to six colours. Accordingly, there is still much room for improvement.GAPDH transcripts from a cell lysate under physiological conditions. At first, they obtained only two GAPDH‐specific RNA fragments. But after complete elimination of RNase H–mediated RNA cleavage, they successfully isolated intact GAPDH mRNA and observed that in the absence of a PAMmer, GAPDH mRNA can still be isolated though with lower efficacy. These data indicate that the Cas9/gRNA complex binds to GAPDH mRNA through direct RNA–RNA hybridization. These results mean that this approach can help to purify endogenous untagged RNA transcripts.One study indicates that the HNH nuclease domain of Cas9 is homologous to other HNH domains that cleave RNA substrates.S. pyogenes Cas9 mutant (dCas9) to deploy a large RNA cargo to targeted DNA loci by incorporating the cargo into the sgRNA. In other words, the sgRNA became a longer sgRNA that contains a sequence of interest such as artificial aptamers, pools of random sequences, and natural long non‐coding RNAs. The sgRNA may bind to genomic loci so that finally we could find out where the RNA sequence of interest is localized. The limitation of this method is that the length of inserted RNA should be at least 4.8 kb. This approach is different from the procedure for inserting the RNA sequence into sgRNA to display RNA domains. The Doudna lab quickly demonstrated that nuclease‐inactive CRISPR/Cas9 can bind RNA in a nucleic‐acid–programmed manner and allows for endogenous RNA tracking in live cellsThe study on CRISPR/Cas9 targeting of RNA began to gain popularity after Doudna's study. David M. Shechner3It is well known that CRISPR/Cas9 has high efficacy of site‐specific gene targeting, but its potential off‐target effects have raised major concerns regarding its application in many respects. What's more, in Luhan Yang et alKRAS, PIK3CA and IDH1 in human cancer cell lines by means of the activity‐decreased RGENs. Moreover, they can also detect point mutations in the BRAF and NRAS genes using RGENs that recognize the “NAG” PAM sequence.Techniques have been developed to detect mutations, for example, Surveyor nuclease assays and T7 endonuclease 1 (T7E1) assays. Both are enzyme mismatch cleavage assays for detection of a single‐base mismatch or small indels and target and cleave mismatched heteroduplex dsDNA. Then, the digested DNA fragments can be distinguished by agarose gel electrophoresis to determine whether there is a mutation. Nonetheless, the limitation of the two assays is that they cannot differentiate homozygous biallelic mutant clones from wild‐type clones and heterozygous monoallelic mutant clones from heterozygous biallelic mutant clones. Jong Min Kim et alKRT12 that causes Meesmann's epithelial corneal dystrophy and can lead to the occurrence of a novel PAM. This meant that they could design a sgRNA complementary to the sequence adjacent to this mutation‐derived PAM to target and cleave the mutated gene. They took advantage of this approach in treating Meesmann's epithelial corneal dystrophy. Another teamKRAS gene to solve the problem of acquired drug resistance to a MEK signalling inhibitor. Both confirmed that the mutation‐ or SNP‐derived PAM could be put to good use in the treatment of some dominantly inherited diseases because the sgRNA/Cas9 complex can specifically disrupt the mutant allele, whereas expression of the wild‐type allele would remain unaffected.It is not easy to control the recognition and cleavage of the RNA‐guided Cas9 nuclease because its specificity is dependent on the length of the sgRNAKRAS, PIK3CA and IDH1 in human cancer cell lines have been identified. As for the novel PAM, the cleavage activity of Cas9 is often employed to disrupt the mutated gene while the wild‐type allele is unaffected. This method is more likely to cure some heterozygous mutations causing an autosomal dominant (AD) single‐allele disease because of the subsequent homology‐directed repair after cleavage by Cas9.Let us review the two assays that are aimed at recognizing an SNV that happened in a genome. An SNV generally has two kinds of consequences: one is the appearance of a new target gene that is different from the former target gene by a single base (relevant to the high specificity of the modified sgRNA/Cas9 complex). The other is the emergence of a novel PAM of Cas9 if there is an ‘‘NGG’’ sequence resulting from the SNV. As for the new target gene, researchers find that by introduction of a single‐base mismatch into the guide RNA, the cleavage specificity of Cas9 can be improved probably because of the decreased enzymatic activity of RGEN, which is quite interesting and needs to be clarified. This assay could be used to distinguish between a mutant type and wild‐type, and thus, three recurrent oncogenic point mutations in genes 4Emerging infectious diseases, such as Zika virus (ZIKV) infections in Latin America and Southeast Asia, have rapidly drawn attention to the need to develop simplified diagnostic tests for clinical practice in some underdeveloped geographic areas. Some authors have published papers about applying the CRISPR/Cas system to the molecular diagnosis to detect the presence of virus such as Zika virus. Keith PardeeStaphylococcus aureus (MRSA) using a CRISPR‐associated protein 9/single‐guide RNA (dCas9/sgRNA) complex as a targeting material to capture genomic DNA of MRSA by specific recognition of the mecA gene (which is implicated in the resistance to methicillin in MRSAS. aureus (MSSA) strains does not contain the mecA gene, the well‐designed dCas9/sgRNA complex should not affect MSSA. Next, the MSSA and MRSA could be separated by Ni‐NTA magnetic nanobeads attached to dCas9 utilizing a magnet. Finally, SYBR Green I (SG I) is added to stain dsDNA and the fluorescence intensity can reflect the concentration of MRSA. It was also confirmed that test samples can be cell lysates without isolation of genomic DNA, this method does not require PCR and therefore is practical and rapid. On the other hand, some researchers have already found a new mecA homologue (mecALGA251) sharing only 70% nucleotide homology with mecA,mecA, and there is a spot of MSSA strains that contains the mecA genemecA gene not specific to MRSA.In contrast, Deng Wulan et alAfter the DoudnaThe principles of the three methods are quite different and all three involve a combination of the CRISPR/Cas or modified CRISPR/Cas system with some other technique to achieve a detection goal. The main features of the three methods are summarized in Table 5Despite the great achievements in CRISPR/Cas technology, there are still many hurdles and limitations in its application of molecular detection. Firstly, the proper recognition of a target site by the sgRNA requires a PAM sequence6Since the discovery of the CRISPR/Cas system, it has been widely used for genomic editing to treat some mutation‐induced diseases. Nevertheless, there is still too much controversy regarding the applications of CRISPR/Cas9 in medical treatments due to the risk of “off‐target” effects. From Yanfang Fu'sThe authors declare no conflict of interests.


How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 , which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We establish an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, allows the prediction of IMP3 targets, and provides a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation. Multidomain RNA-binding proteins recognize specific target sequences through mechanisms that are not well understood. Here the authors present an integrated approach to define the RNA-binding specificity and RNP topology and apply it to the analysis of the prototypical multidomain RNA-binding protein IMP3. The three mammalian paralogs are often described as oncofetal due to their expression primarily during embryogenesis and severe phenotypes in the case of impaired expression3.The insulin-like growth factor 2 mRNA-binding protein 3 (IMP3 or IGF2BP3) belongs to a family of three highly conserved RNA-binding proteins  that are involved in post-transcriptional gene regulation of mRNAs4. This caging of mRNAs ranges in its functional spectrum from packaging for cytoplasmic transport5, delayed translation within stable mRNPs8, cytoplasmic storage, and protection against premature miRNA-directed mRNA regulation12. Several target mRNAs have been suggested13, with IMP1 associating with the ACTB mRNA zipcode element and all three IMPs regulating HMGA2 stability via the 3′-UTR as the currently best-studied examples16.The currently best-understood IMP-mediated mechanism of modulating mRNA fate comprises the so-called safe housing of specific transcripts in mRNP granules19.In contrast to IMP1 and IMP2, the biological relevance of IMP3 has long been underestimated. Research on IMP3 largely focused on its association with many cancer-related tumor entities, since its re-expression correlates with a poor prognosis for patients, classifying IMP3 as a tumor marker1. It has been a long-standing question how multiple RBDs cooperate in specific and high-affinity RNA-target recognition: Which of the individual domains are involved, what are their contributions, and how flexible is the RNA–protein interaction pattern?The IMP protein family represents a prototypical example of multidomain RBPs and is characterized by a common architecture of six potential RNA-binding units: two N-terminal RNA-recognition motifs (RRMs) and four consecutive hnRNP K-homology (KH) domains24. For the IMPs, structural information is available only for single RRMs of IMP2  and IMP3 . The presence of a very short linker sequence suggests that the two domains are arranged in a compact tandem, which might drive their RNA specificity. Analogously, there is evidence that the KH1–2 and KH3–4 tandem domains represent prearranged RNA-binding modules for recognition of bipartite RNA sequence motifs. Structures of the human IMP1 KH3–414, as well as the KH3–4 di-domains of the chicken ortholog ZBP116 proved the existence of an extended domain interface between KH3 and 4. These structures suggest target RNA motifs to require a minimal spacing to be recognized by the tandem RBDs. For example, KH3–4 of ZBP1/IMP1 recognizes a combination of two sequence elements: CGGAC-N10–25-(C/A–CA–C/U) in both possible arrangements16.Assessing the contributions and cooperativity of multiple RBDs in binding to multipartite RNA motifs is challenging, and a generally applicable approach has not been described so far. Due to the potential dynamic domain arrangements of multiple RBDs, structural studies require an integrated approach, combining solution techniques and crystallography25 and in vitro selections 28, all suggesting an overall CA-rich consensus. However, the major limitation of in vitro selection approaches is that they usually start with short degenerate sequences, which can accommodate only a single RNA-binding motif. Therefore, the contributions of individual domains have remained elusive. Finally, while previous studies provide evidence for an essential role for KH domains in RNA interaction, no function had been ascribed yet to the two RRMs30.Previous studies proposed short recognition sequences of IMPs, based on in vivo CLIP40) as a basis for SELEX, to allow multiple RNA contacts with more than a single RNA-binding domain, and a corresponding bioinformatic spacing analysis. This led us to the discovery that IMP3 recognizes—through the activity of all of its tandem RNA-binding domains—an extended array of multiple cis-acting RNA elements, composed of CA-rich motifs and sequences with a common GGC core. These biochemical findings are supported by integrated structural biology, combining crystallography and NMR for structural analysis and RNA-binding studies of IMP3 KH and RRM-tandem domains.To study IMP3 as a prototypical example of a multidomain RBP, we established a systematic, domain-resolved SELEX procedure coupled with RNA-seq and combinatorial bioinformatic approaches. Importantly, we used a very long degenerate sequence . Therefore, we relied on truncated tandem domains for our analyses: RRM1–2, KH1–2, KH3–4, as well as an extended version containing all four KH domains, KH1–4 , which are bound by the KH tandem domains. Besides that, we provide evidence that the RRM1–2 domains contribute additional binding of a CA-rich element.z-score analysis , or a combination of a CA-rich motif with one of the identified GGC-core elements. For all combinations , we observed shorter spacing of N2–11 nucleotides, with a maximum at N4–6. However, longer spacing was found to be clearly specific for either one of the two very similar GGC elements (GGCA versus CGGC): Only GGCA-N18–21-CA or CA-N22–25-CGGC were enriched, but not the respective reverse orientations , and the respective combination with CA-rich motifs identified through analysis of the full-length protein . This observation is supported by the results obtained for the full-length IMP3 protein , most likely representing a 3′-CA extension of these motifs rather than real spacing, since previously published data argue for a minimal spacing requirement of N10–25 between two motifs recognized by a KH di-domain.Finally, for KH1–4, we detected a mix of enriched motif spacing already observed for the separate KH1–2 and KH3–4 domains, with a preference for both 2–11 for GGC and CA elements in both orientations within the full-length context of all six RBDs (FL-IMP3). While a mixture of spacing/orientations for all domains is expected, a comparison with KH1–4 argues that specifically shorter spacing reflects the influence of RRM1–2. Therefore, we interpret this as spacing between a GGC motif bound by one of the KH domains and a nearby CA element recognized by RRM1–2.In addition, spacing analysis for RRM1–2 revealed strong enrichment for CA-rich motif combinations in all positions within the 25- nts window, but not for the GGC-core elements Fig. , middle,Based on these datasets, we assembled a working model of how IMP3 recognizes RNA Fig. . Due to GGCA-N20-CACA-N14-CACA-N22-CGGC-N4-(CA)4 4 Fig. .Fig. 3Va32P-labeled 101-mer RNA with high affinity  revealed that the full-length protein recognizes the To test for motif contribution within the 101-mer sequence, we either substituted the CA motifs (CA->UG), the GGC-core elements (GGC->UG), or a combination of both , each by mutating to UG . Surprisingly, the binding affinity remained unchanged, suggesting that either this element does not significantly contribute to the overall affinity or that IMP3 can recognize the element in both positions, consistent with our spacing analysis , resulting in a four-fold decrease in affinity Fig. . The addTo address the stoichiometry of the major RNA–protein complex observed here and in the following assays, we also compared complex formation with full-length IMP3 proteins with or without GST tag, as well as with an equimolar mixture of both of them , their RNA binding was inactivated by mutation (GKEG motif to GDDG), while maintaining the crucial tandem context16, resulting in four possible combinations RMs Fig. . The majRMs Fig. . Interes3 6-mer RNA that should not extend toward RRM2 in the tandem domain arrangement. However, we found almost identical CSPs   Fig. . The mod5 revealed a low-micromolar affinity, and in line with our NMR data, we found the same affinity for RRM1–2 when binding to the 6-mer CA-RNA , indicating that this domain indeed contributes to RNA binding also in the full-length context.To further verify the suggested concept with all IMP3 RBDs engaged in multivalent RNA recognition, we next tested the contribution of individual tandem domains within the full-length-protein context. Therefore, we mutated critical amino acids in the respective domains to inactivate individual tandem domains . Such a correlation approach may also be valuable to predict IMP3 targets.RPL32, as a negative control, with a very low B index (0.01) and containing no motif array; SLC6A14 and UHMK1 as two examples of predicted IMP3 targets ; and HMGA2, a known IMP3 target (B index: 1.58). In addition, we had previously identified and validated ANKRD17 exon 29 as an IMP3 target that is not only spliced in the canonical mRNA, but also additionally processed into a circular RNA27. Analysis of the sequence and iCLIP-tag counts also predicted ANKRD17 exon 29 as an IMP3 target (B index: 0.16), with one of the motif arrays exhibiting a motif-spacing pattern very similar to our rationally designed 101-mer RNA (see bottom panel).Using HepG2 whole-cell polyA+RNA-seq data (ENCODE/CSHL) as an expression reference, iCLIP-tag counts in each 3′-UTR, normalized by the respective expression levels, were summarized to yield an index of in vivo binding (B index). Figure On a global level, from the total set of 11,084 3′-UTRs expressed in HepG2 cells, 4834 (44%) contain no motif array, 1747 (16%) contain one, 1825 (16%) two or three, and 2678 (24%) at least four arrays Fig. . The disSLC6A14 and UHMK1) were positively validated . This was further validated by quantitative EMSA with an isolated region from the 3′-UTR of the well-studied IGF2 mRNA  into a luciferase reporter construct and measured the effect of IMP3 motif mutations, let-7 seed mutations11, and a combination of both on relative luciferase activity , indicating functional inactivation of the IMP3-binding site , independent of the IMP3 expression status.In comparison with the WT ite Fig. . In contHMGA2 expression are in fact due to changes in IMP3-binding affinity, we performed quantitative EMSAs , higher-order complexes could be detected, indicating multiple IMP3 molecules bound to this RNA.To confirm that the observed regulatory effects on SAs Fig. . WhereasHMGA2 3′-UTR that contains miRNA-binding sites in close proximity, thereby protecting the mRNA from let-7-mediated repression.Taken together, our in-depth analysis of sequence requirements for IMP3–RNA interaction and the functional validation supports the suggested safe-housing mechanism: Through sequence-specific formation of RNP complexes, IMP3 shields a specific region within the 28, systematic experimental approaches to study combinatorial RNA recognition by multidomain RNA-binding proteins have not been reported so far. Also, commonly employed global approaches to map protein–RNA interactions, such as CLIP, RIP, and RNACompete, have been analyzed with the aim to reveal short consensus sequences. Thereby, the systematic description of multidomain RBPs as well as rational searches for high-confidence and functional target sequences were severely limited20.Members of the IMP protein family are prime examples for multidomain RBPs, where both affinity and specificity are achieved through simultaneous engagement of multiple domains with their respective RNA elements. Although bioinformatic analyses can predict some features of RNA recognition by multidomain proteins30. Our SELEX approach based on a N40-degenerate sequence revealed that the KH domains recognize two different types of RNA motifs: CA-rich motifs and elements with a common GGC core. We note that choosing 40 degenerate positions for our SELEX analysis limits the analysis of motif combinations, yet allows the enrichment and spacing analysis for at least two adjacent motifs, so that combined with the parallel study of various subdomains, a model for RNA binding of full-length IMP3 with its six RNA-binding modules that span ~100 nts could be derived. Structural analysis of the KH1–2 and RRM1–2 tandem domains and mapping of RNA interactions by NMR corroborated the specific interaction between subdomains and SELEX-derived RNA motifs. ITC clearly proved a cooperative interaction of tandem KH1–2 with a properly spaced, bipartite RNA motif. Our data suggest that in complex with KH1–2—similar to the situation with KH3–4— the RNA adopts a looped conformation that fits the narrow window for linker length between motifs.Here, we focused on IMP3 to dissect its complex RNA binding through a systematic SELEX-seq approach: We found that all di-domains  were active in RNA binding, while most previous studies had argued that only the KH domains 3 and 4 guide RNA recognitionGGAC motif, we find that IMP3 recognizes two related GGC-core elements (GGCA and CGGC), including their relative arrangement in combination with an additional CA-rich motif. Therefore, our data argue for KH1–2 and KH3–4 acting as independent tandems, both recognizing a combination of one CA-rich motif and one GGC element, with KH1 and KH4 binding the respective GGC elements.In contrast to the IMP1-associated CSpecifically, for KH3–4, and to a lesser extent for KH1–2 and KH1–4, we also observed an enrichment of AU-rich sequences. However, these sequences were underrepresented in full-length IMP3. This may reflect unspecific binding caused by protein truncation. Indeed, C-terminally shortened variants of KH3–4 and KH1–4 were diminished in RNA binding. To experimentally test the contribution of AU-rich sequences to IMP3–RNA binding, we quantitatively assayed RNA binding of full-length IMP3 protein to an additional mutant derivative of the 101-mer RNA, CA->UA, where both central CA elements were changed to AU . In fact, the consensus sequence bound to IMP3 with a high affinity, depending on the presence of the individual sequence elements, and involving all tandem RBDs. Importantly, our consensus motif array also allowed the successful prediction of IMP3 mRNA targets  was detected for KH1–2 within the full-length IMP3 protein, indicating a restricted flexibility of the domains in their canonical context. This is further reflected by a decreased affinity when the order of KH1–2 RNA elements is swapped within the 101-mer RNA. The proposed topology of RNA elements and RBDs may be induced and stabilized additionally by the kinetic rates of binding, as suggested by Ramos and co-workers for looped RNA around KH3–4 at in vivo concentrations16. Notably, the NMR data of KH1–2 with a corresponding 34-mer RNA ligand show significant line broadening that is primarily caused by the increase in molecular weight.We observed that isolated tandem domains  seem to tolerate the enriched motif combinations in both possible arrangements, a phenomenon that was previously described for KH3–4 of IMP130. This greatly exceeds previous studies, including large-scale surveys of many RNA-binding proteins28, which for the most part were restricted to short recognition sequences. These may even be misleading in many cases, since only particularly dominant sequence elements are usually identified by these approaches. Considering that most RBPs belong to the multidomain type37, our approach presented here on the IMP3 example should advance our understanding of clustered target RNAs41, and should help in global rational searches for functional target sites as well as in future engineering of tailored multidomain RBPs42.In summary, we provide the first domain-resolved insight into the complex process of IMP3–RNA recognition through concerted interaction of multiple, clustered RNA sequence elements and all RBDs of IMP3. Multivalent interactions of individual domains, each with limited specificity, cooperatively add up to the very specific engagement of full-length protein with target RNAs6-tag and TEV-cleavage site, and were cloned into the pGEX-6P2 expression vector . The GST–IMP3–TEV–His fusion proteins were purified via Ni2+-agarose beads (Qiagen), and the His6-tag was subsequently cleaved off through TEV protease according to the manufacturer’s instructions (Life Technologies). All protein preparations were dialyzed  and stored at −80 °C. IMP3 RNA-binding domain mutants were produced by PCR mutagenesis, using the Q5 Site-Directed Mutagenesis Kit following the manufacturer's instructions (NEB).The full-length (FL) and truncated IMP3 derivatives used for SELEX experiments were ordered as codon-optimized DNA fragments encoding FL-IMP3 (Met1–Lys579), RRM1–2 (Met1–Asn163), KH1–2 (Pro164–Phe376), KH3–4 (Pro377–Lys579), and KH1–4 (Pro164–Lys579) (ThermoFisher), with additional HisE. coli. The Δ versions of KH1–243 were created by restriction-free site-directed mutagenesis. Proteins were expressed as thioredoxin fusion proteins comprising an N-terminal His6-tag and a TEV-cleavage site between thioredoxin and the gene of interest in the pETTrx1a vector . RRM1–2 was expressed by inoculating an LB overnight culture with a clone from a freshly prepared BL21 (DE3) LB culture plate supplemented with 0.35 mg/ml kanamycin. The culture was diluted into the medium of interest and grown to an OD600 of ~0.8 before induction with 0.5 mM IPTG. Cells were then grown for another 4–6 h at 37 °C before harvesting. Pellets were resuspended in lysis buffer , incubated on ice for 30 min, and sonicated. Cleared lysates were subjected to Ni2+-agarose beads. After intensive washing, beads were incubated with 500 µg/l culture of TEV protease in lysis buffer for 3 h with gentle shaking at room temperature. Subsequently, the bead supernatant was collected, concentrated, and gel-filtrated in 20 mM Bis-Tris, 500 mM NaCl, and 2 mM TCEP, pH 6.5. The respective protein–monomer peak was pooled and salt concentration was adjusted to 150 mM. For RRM1–2, we included an additional ion-exchange chromatography step to reduce the level of nucleic acid contaminations. This was carried out on a 5/5 MonoS cation exchange column , running a gradient from 50 to 1000 mM sodium chloride in 20 mM Bis-Tris and 2 mM TCEP, pH 6.5. Fractions of intact protein were pooled and dialyzed against the final buffer as before.For structural studies, RRM1–2 (Lys2–Asp156) and KH1–2 (Lys192–Ile355) tandem-domain expression constructs were cloned from the human IMP3 full-length protein sequence optimized for expression in 40) was prepared by T7 transcription. In total, 40 pmol of full-length GST–IMP3 truncated derivatives, or GST alone (as negative control), were used for four rounds of selection with 4 nmol of SLX-N40 transcript. The stringency of washing steps  was increased for each round of selection . SELEX selections were carried out with the fusion proteins bound to glutathione–sepharose . RNA aliquots from each round were used for barcoding by reverse transcription with the SLX_RX reverse primers. cDNA libraries were amplified by PCR . The final library pool was subjected to high-throughput sequencing on a MiSeq platform . PhiX control library was added to increase sample complexity (Illumina). For primer sequences, see Supplementary Data The RNA pool with a degenerate sequence of 40 nucleotides  containing two tetramers with a spacing of 0–25 nts were summed up, and the CA-rich refers to tetramers containing at least three C or A residues, alternating at least once, and excluding CCC, AAA, and any G nucleotides.z-score mean values for individual positions (0–25 nts) were assigned and represented as a heatmap in Fig. For each group, the 27 are available from the Sequence Read Archive (SRA) of NCBI (SRP139915).Sequencing data for the IMP3 iCLIP in HepG2The annotated 3′-UTRs (Comprehensive Gene Annotation Set from GENCODE version 19) were selected to evaluate IMP3 in vivo RNA-binding efficiency, with HepG2 whole-cell polyA+RNA-seq data from ENCODE/CSHL (genome.ucsc.edu) applied as a RNA expression reference. A binding index (B index) of each in HepG2 cells expressed 3′-UTR was calculated as the ratio of iCLIP-tag counts and the expression level estimated by RNA-seq read coverage.GGC or GGCA), with a spacing of 30–200 nt, (GGC) - N30–200 - (GGC).Two flanking GGC-core elements (C≥10 - (CA) - N≥6 - (CA) - N≥10 - (GGC).Two CA elements, out of the 13 tetramer CA elements used for spacing analysis of 4-mer motif combinations (Supplementary Data 6–200 - (CA) or (CA) - N6–200 - (GGC).Downstream or upstream of the two flanking GGC-core elements, a CA motif within a distance of 6–200 nt, (GGC) - NTo identify SELEX-derived motif arrays, 3′-UTR sequences were screened by sequentially applying the following criteria , allowi5, (CA)3, (GU)5, the KH1–2-related GGC and CA 7-mers, and the 34-mer were obtained from IBA (Göttingen) or Eurofins (Ebersberg). Lyophilized RNAs were dissolved in nuclease-free water, heated to 95 °C for 5 min, snap-cooled, aliquoted, and stored at −80 °C.The RRM1–2-related RNAs (CA)XDS44 and scaled using SCALA45. Intensities were converted into structure-factor amplitudes using the program TRUNCATE46. Supplementary Table The crystallization experiments for IMP3 KH1–Δ2 domain were performed at the X-ray Crystallography Platform at Helmholtz Zentrum München. Initial screening was done at 292 K, using 12 mg/ml of protein with a nanodrop dispenser in sitting-drop 96-well plates and commercial screens. Crystals appeared after 1–2 days with sufficient size for X-ray diffraction experiments. The best dataset was collected for a crystal grown in 0.08 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, and 30% w/v polyethylene glycol 4000 (Hampton Research NATRIX screen). For the X-ray diffraction experiments, the crystals were mounted in a nylon fiber loop and flash-cooled to 100 K in liquid nitrogen. Prior to freezing, the crystals were protected with 25% (v/v) ethylene glycol. Diffraction data were collected at 100 K on the PX X06SA beamline . The diffraction data were indexed and integrated using Auto-Rickshaw pipeline47. Three-dimensional model of KH1–2 domains of the neuronal splicing factor Nova-1 (PDB-ID: 2ann)49 was used as a search model. For the molecular replacement step followed by several cycles of automated model building and refinement, the Auto-Rickshaw pipeline involved the following X-ray crystallography software: MORDA, CCP450, SHELXE51, BUCCANEER52, RESOLVE53, REFMAC554, and PHENIX55. Model rebuilding was performed in COOT56. The further refinement was done in REFMAC554 using the maximum-likelihood target function. The stereochemical analysis of the final model was done in PROCHECK57 and MolProbity58. The final model is characterized by R/Rfree factors of 23.29/29.27% (Supplementary Table The structure of KH1–2 domains was solved by the 15N ammonium chloride (titrations and relaxation experiments) and 2 mg/ml 13C glucose (triple-resonance experiments for backbone assignments). Wild-type KH1–2 has additionally been expressed in 99.5% D2O, following a previously described protocol59. Briefly, cells were grown in sequential steps of 0% D2O and 2 g/l glucose (50 ml), 50% D2O and 2 g/l glucose (250 ml), and finally 99.5% D2O and 2 g/l 2H;13C glucose (2 l) with full transfer of cell mass between steps. All experiments were performed in 20 mM Bis-Tris, 150 mM NaCl, 2 mM TCEP, 0.02% sodium azide, and 5–10% of D2O. NMR backbone assignments have been obtained using the following experiments: HNCA, HNcoCA, HNCACB, CBCAcoNH, HNCO, HNcaCO, and 15N-edited NOESYs. All datasets were acquired from Bruker Avance spectrometers of 600–950-MHz proton frequency equipped with triple-resonance cryo-probes using Topspin 3.2. The data were processed with Topspin and analyzed using the CCPNMR Analysis software package60 and SPARKY61. Sample concentrations were 250–650 µM.For NMR measurements, proteins were expressed in M9 media supplemented with 0.5 mg/ml 15N autorelaxation and {1H}–15N heteronuclear NOE data were recorded with a 300 µM sample using pseudo-3D experiments with the delays 4, 8, 16, 32, 48, 64, 96, 128, 192, 256, 512, and 1024 ms for R1 and the delays 5, 10, 20, 40, 60, 80, 100, 125, 150, 200, and 300 ms for R2. Peak intensities were fitted and plotted with Analysis. τC was calculated based on the ratio of R1 and R2. NMR titrations of KH1–2 versions and RRM1–2 were performed with samples of 50–100 µM protein by adding the denoted stoichiometries of RNA from a 4 mM stock solution. Combined chemical shift perturbations were calculated using the formula Δδ = [6(Δ1H)2 + (Δ15N)2]0.5. All NMR experiments were carried out at 25 °C.RRM1–2 Omnisec device with an integrated sample changer and equipped with a semianalytical SD200 10/300 Superdex column (GE). Samples of RRM1–2 had concentrations as indicated; the used sample volume was 125 µl. Runs were performed in buffers as for NMR, but no D2O. UV (260 and 280 nm), right-angle light-scattering and refractive index data were analyzed using the integrated Omnisec software, and molecular weights were determined using a dn/dc value of 0.185 for protein. Therefore, peak picking and baseline definition were performed automatically or manually. The system was calibrated with 5 mg/ml bovine serum albumin (66.5 kDa) as a standard.SLS runs were performed on a Malvern 62 package version 2.8, including the plot of paired-distance distribution, P(r), the determination of Dmax and Rg, and the calculation of Porod volumes and molecular weights with DATPOROD. Theoretical scattering curves derived from the KH1–2 crystal structure or RRM1–2 models were calculated with Crysol63.SAXS experiments were performed in-house or on beamline BM29 at ESRF, Grenoble, France. Sample concentrations were 1–7 mg/ml. Reference runs in buffers were performed multiple times and used for buffer subtractions. Measurements were carried out as technical triplicates in four to ten frames to enable the exclusion of data in the case of radiation damage. Data were processed and analyzed with the ATSASEOM264 and fitted against the SAXS scattering curve at the highest concentration. We obtained an ensemble of four structures with populations of 60, 20, and two times 10% that showed a χ2 fit of 1.335. We chose the highest-populated structure, that also represented the most compact moiety (Dmax of 61 Å) and used it to include the following restraints: The 7-mer linker (residues 73–79) was rationally probed for possible conformations, i.e., the minimum distance between residues 72 and 80 in a U-turn loop (6 Å), within a α-helix (12 Å) or the maximum distance when arranged in a β-strand (26 Å). The first would have led to steric clashes between RRM1 and 2, and since our secondary chemical shift data did not reveal a clear preference for α-helical or β-strand elements, we set the distance to be 16 Å. That allows for sufficient flexibility but would still be in line with a high degree of rigidity (see heteronuclear NOE and relaxation data) and fulfills the obtained Dmax of 54 Å when manually arranging RRM1 and 2. In order to satisfy CSPs, we included a maximum distance of 30 Å between residues Val35  and Ser127 (RRM2). The latter—despite nonfunctional RNPs in RRM2—still significantly senses the binding of (CA)3 RNA, which would approximately comprise a maximum extension of 30 Å. Finally, the relative twist of RRM1 versus RRM2 around the positively charged inter-domain linker was limited, given the fact that it senses strong CSPs (see Lys77), indicating that it could be arranged along with the RNA. As such, we decided to prevent a cross-brace possibility for a linker and RNA and suggest the RNA to bind along the RRM1 β-sheet and the linker, thereby indirectly interacting with Ser 127/128. Hence, we put a 15-Å distance between the strongly shifting residue Thr115 and Glu55 to impair the free rotation of domains. All two-domain models were used in the program Coral65 and fitted against the scattering curves, until the crucial parameters Dmax, RG, and Porod volume were optimized and the model was approximately in line with the CSP plot. The final model showed a χ2 of 1.9, as given in Supplementary Fig. Due to the lack of an experimental structure of RRM1–2, we used SAXS data to filter randomized tandem arrangements. Therefore, RRM1 was modeled based on the IMP2 RRM1 NMR structure (PDB-ID: 2cqh), including residues 1–72. For the RRM2, we used the available structure (PDB-ID: 2e44) and adjusted the domain boundaries to residues 80–156. This fragment was in perfect fit with a CS-Rosetta-based structure based on our backbone NMR data. The linker region 73–79 was kept flexible and the two domains used as an ensemble in 10,000 random starting structures were generated with ITC measurements were performed with a MicroCal PEAQ-ITC device  in the NMR buffer. In all experiments, RNA was titrated from a stock of 10–20-fold concentration excess to 20–40 µM protein provided in the reaction cell. In a standard ITC run, we used 19 injections of 2 µl with 150-s spacing at room temperature with a 750-rpm stirring speed. Raw data were analyzed with the integrated analysis tool and heat production was fitted to a one-site binding model. Where appropriate we performed a buffer subtraction.32P-UTP-labeled by T7 transcription from annealed oligo cassettes. SELEX-motif-containing regions of IGF2 (NM_001007139.5), HMGA2 (NM_003483.4), and ANKRD17 (NM_032217.4) transcripts were PCR amplified and used for T7 transcription and labeling (sequences given in Supplementary Data 32P-UTP-labeled RNA (5 nM) in a final volume of 10 µl. The reaction was first incubated for 30 min at room temperature, and then placed on ice for 5 min. Each sample was supplemented with loading buffer , and loaded onto a cold native 5% TBE gel (containing 5% glycerol) that had been pre-run for 30 min. Electrophoresis was performed for 50 min with 45 mA at 4 °C. Complexed and free RNA was visualized for quantitation by the Typhoon FLA 9500 Phosphorimager system . Curve fitting of raw data using the quadratic binding equation66 or the Hill equation . Whenever source data are available, this is indicated within the respective figure legends.RNAs of the 101-mer series were produced and 67. For pulldown of IMP3 from HeLa cell lysate, 2.5 × 106 cells were lysed in lysis buffer  and incubated with 40 pmol of 3′-biotinylated RNA bound to NeutrAvidin agarose beads (ThermoFisher) in a total volume of 200 µl for 30 min at room temperature. Pulldown of recombinant IMP3 was performed by incubation of 10 pmol 3′-biotinylated RNA bound to NeutrAvidin agarose beads (ThermoFisher) with 1 pmol protein in binding buffer  in a total volume of 200 µl for 30 min at room temperature. After three washing steps with washing buffer , bound protein was released in SDS-sample buffer  and heat denaturation at 95 °C for 10 min. Samples (10% input and 50% pulldown) were analyzed by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide gel) and Western blotting with polyclonal anti-IMP3 antibody (Millipore) against endogenous IMP3, or anti-GST antibody (Pharmacia Biotech) against the recombinant and GST-tagged IMP3 version.RNAs of the 101-mer series were produced by T7 transcription  from annealed oligo cassettes and chemically modified by 3′-biotinylationCell lysates were prepared in RIPA buffer . Antibody binding was performed for 2 h at 4 °C, using a polyclonal anti-IMP3 antibody (Millipore) and as a mock control, anti-FLAG antibody (Sigma-Aldrich). Bead capturing was carried out for 1 h at 4 °C with protein-G dynabeads (Life Technologies), and protein–RNA complexes were washed , increasing the stringency during the washing steps. RNA from the input and from the immunoprecipitated fractions was extracted by TRIzol (Ambion), followed by ethanol precipitation and reverse transcription . Real-time PCR was carried out using Luna Universal qPCR Master Mix (NEB) and an Eppendorf realplex thermocycler. The fraction of bound target RNAs in RIP assays was calculated with each target normalized to the corresponding input fraction (results represented as percent of the input). Biological triplicates were used to calculate standard deviations. For primer sequences, see Supplementary Data For the CRISPR/Cas9-mediated genomic deletion of IMP3, ES-2 cells were transfected with two CRISPR guide RNAs  and Cas9 nuclease (pcDNA_Cas9_T2A_GFP), using Lipofectamine2000 (ThermoFisher) according to the manufacturer’s protocol. Single-cell clones were generated by seeding one RFP- and GFP-positive cell per well using flow cytometry (BD FACSAria II). The deletion of IMP3 was validated by Western blotting using paralog-specific anti-IMP3 antibodies . CRISPR guide RNAs are summarized in Supplementary Data HMGA2 3′-UTR (NM_003483.4) containing the IMP3 SELEX-derived motif and the let-7 seed sequences, together with the respective mutants , were ordered as DNA fragments (Supplementary Data 5 ES-2 cells (with or without genomic IMP3-KO) were seeded per well, in a 12-well plate. Cells were transfected with 250 ng of plasmid DNA and 4 µl of Turbofect (ThermoFisher), and incubated for 24 h. After three washing steps with 1x PBS (Gibco), cells were lysed in 250 µl of 1x Lysis-Juice (PJK) according to the manufacturer’s instructions. Luminescence was monitored for Firefly luciferase, using the Beetle-Juice Kit, and for Renilla luciferase, using the Renilla-Juice Kit (both PJK) with a Centro LB 960 Luminometer (Berthold Technologies). Relative luciferase activities were calculated as a ratio of Firefly and Renilla raw values with three technical replicates per sample and a total of three independent biological replicates. ES-2 cells with or without IMP3-KO were characterized by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide gel) and western blotting with antibodies specific for IMP1 , IMP2 , IMP3 (Millipore), and GAPDH as a negative loading control (Sigma). The region of the Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Source Data


Trypanosoma brucei, are transcribed individually. Also unlike most mitochondrial mRNAs, the 5′ end of these transcripts harbor a triphosphate that is hydrolyzed. This modification is carried out by a putative Nudix hydrolase called MERS1. The Nudix motif in MERS1 is degenerate, lacking a conserved glutamic acid, thus it is unclear how it may bind its substrates and whether it contains a Nudix fold. To obtain insight into this unusual hydrolase, we determined structures of apo, GTP-bound and RNA-bound T. brucei MERS1 to 2.30 Å, 2.45 Å, and 2.60 Å, respectively. The MERS1 structure has a unique fold that indeed contains a Nudix motif. The nucleotide bound structures combined with binding studies reveal that MERS1 shows preference for RNA sequences with a central guanine repeat which it binds in a single-stranded conformation. The apo MERS1 structure indicates that a significant portion of its nucleotide binding site folds upon substrate binding. Finally, a potential interaction region for a binding partner, MERS2, that activates MERS1 was identified. The MERS2-like peptide inserts a glutamate near the missing Nudix acidic residue in the RNA binding pocket, suggesting how the enzyme may be activated. Thus, the combined studies reveal insight into the structure and enzyme properties of MERS1 and its substrate-binding activities.Most mitochondrial mRNAs are transcribed as polycistronic precursors that are cleaved by endonucleases to produce mature mRNA transcripts. However, recent studies have shown that mitochondrial transcripts in the kinetoplastid protozoan,  Trypanosoma brucei, T. cruzi, and Leishmania spp., are named after their unusual mitochondrial DNA called the kinetoplast . MERS1 is a 395 residue protein, MERS2, a 946 residue peptatricopeptide repeat polypeptide and MERS3, a 189 amino acid protein with no known motifs  that forms a loop–helix–loop motif  lacks one of the key metal ion coordinating acidic residues, which in MERS1 is a threonine instead of an acidic residue. Metal coordination by the conserved acidic residues in the Nudix motif is required for catalysis by most Nudix enzymes. However, the mechanisms used by Nudix hydrolases are quite diverse  and crosslinking and affinity purification (CLAP) experiments uncovered a consensus motif of 5′-GAGAGGGGGGUUU-3′ that was associated with MERS1 and MERS2 . HoweverMultiple sequence alignments of MERS1 homologs revealed that the amino-terminal ∼70 residues of these proteins are poorly conserved. This is in contrast to the remainder of the protein sequence, which shows strong conservation among homologs . Data anRwork/Rfree values of 22.9%/25.7% to 2.45 Å resolution  using a selenomethionine-substituted crystal  . The strsolution . The MERsolution A. Domainsolution , 2A. A Nsolution A. The stsolution . Howeversolution B. Interesolution A. The prEscherichia coli Nudix hydrolase YmfB  searches  failed tase YmfB  and E. cydrolase . These sydrolase A. Amino ydrolase A.Supplemental Fig. S1). Interactions to the ribose moiety are made by backbone atoms of residues Tyr275 and Gln276. Finally, there are numerous hydrophobic and stacking interactions from MERS1 residues to the guanine base. These are provided by the side chains of Tyr197, Tyr275, and Val285. In addition, the side chain of Arg284 makes cation-π interactions with the guanine base. These contacts shield the base from the solvent. The guanine base O6 atom makes a hydrogen bond to the amide nitrogen of MERS1 residue Val285  make hydrophobic contacts to MERS1 Nudix motif residues Trp240 and Leu242 (Data indicate that MERS1 forms a complex with the MERS2 and MER3 proteins and that these interactions activate MERS1 activity . The 946d Leu242 A. The smd Leu242 A. It is M/L)XXALXXEL and probed the MERS2 and MERS3 sequences for similar motifs. While no homology was found between the sequence and that of MERS3, one strong match for the motif was located within the central PPR motif region of MERS2 (residues 394–402). This region is predicted to be helical and contain a PPR motif in MERS2. Attempts to purify MERS2 or a MERS2 truncation containing this PPR motif for binding analyses, however, were unsuccessful. Thus, we obtained a fluoresceinated peptide encompassing this MERS2 region, F-SVSKQAVCLMERHLRRVPGARRNELKVALSKELKER (where underlined residues correspond to the peptide consensus residues) and utilized this peptide in FP binding studies. The experiments revealed that the peptide bound MERS1 saturably with a Kd of 2.5 ± 0.8 µM (PSKPLKER). This peptide showed only weak binding to MERS1  as a search model in molecular replacement (MR). A clear solution was obtained and the structure was refined to final solution . Like thsolution A. This rKd of 40 ± 0.9 nM . In contrast, a DNA site, 5′-GTGAGTACTCAC-3′, showed essentially no binding  and changing the adenine in the second position also had no effect  is ∼15 Å from the Nudix motif and thus too far to be accessible for catalysis by residues in the Nudix motif, suggesting that it may not use a typical Nudix hydrolase type mechanism. Alternatively, the GTP might be bound at a site that does not correspond to the 5′ triphosphate binding pocket. Precedent for the latter possibility comes from studies on bacterial RNA pyrophosphohydrolase (RppH) enzymes . RppH enposition . Also, two steps . Further a dimer . Consistound RNA . Our bioound RNA A. The fi0%/25.1% .Supplemental Fig. S3A). Weak density is present for RNA 5′ to the first constructed nucleotide, which would be close to the Nudix motif, however RNA molecules could not be unambiguously fit into this density . This appears consistent with protein–RNA crystal structures wherein density is generally visible only for tightly bound nucleotides . The two MERS1 subunits in the ASU do not form significant interactions that would be suggestive of a dimer. Nor were any other possible MERS1 oligomers observed due to crystal packing, indicating that MERS1 does not form higher-order oligomers upon RNA binding. The helical interaction derived from crystal packing in the MERS1–GTP complex was also not observed in this structure, which harbors different packing. In the structure, density is evident for four RNA nucleotides bound to each MERS1 subunit A. The foleotides . The eleleotides B. Furtheleotides B. Strikileotides C. The phComparison of the MERS1–RNA and MERS1–GTP structures revealed that RNA binding induces conformational changes in the RNA binding pocket C; SuperiThe MERS1–RNA structure shows that there are a large number of contacts between the RNA and MERS1 D. The phThe MERS1–RNA structure revealed that the RNA binding pocket is particularly rich in arginine residues, which are involved in numerous contacts with the RNA. To test the roles of arginines the structure indicates as important in RNA binding, we generated R199A, R284A, R356A, and R364A substitutions A. The R3E. coli and B. subtilis RppH proteins in which nucleotides that are located 3′ to the 5′ triphosphorylated nucleotide are key for binding specificity and stability  may function in magnesium binding and catalysis  cells were transformed with the expression plasmid and protein expression was induced by addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM for 4 h at 37°C when the cells had reached an A600 of 0.4–0.6. The cells were reconstituted in 25 mM Tris HCl pH 7.5, 300 mM NaCl, 5% glycerol, 0.5 mM β-mercaptoethanol and lysed with a microfluidizer. The expressed MERS1 protein was found in the soluble fraction and was purified using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography. The protein was further purified by Superdex 75 size exclusion column (SEC) chromatography. MERS1 mutants were made using the Quikchange mutagenesis protocol and expressed and purified as per the WT protein.For structural studies, the nucleotides encoding residues 37–395 of MERS1 were cloned into pET15b between the BamHI and NdeI restriction sites. 4. The crystals grew at room temperature and appeared after several weeks. Crystals of MERS1 bound to the RNA sequence, 5′-GAGAGGGGGGUU-3′, were obtained by mixing the protein (10 mg/mL) with twofold excess RNA and then concentrating the mixture further in microcons (with 3 kDa molecular weight cutoff). The complex was concentrated threefold and then mixed 1:1 with a crystallization reservoir consisting of 0.1 M N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) pH 10.5, 800 mM potassium phosphate dibasic, 1.2 M sodium phosphate monobasic, 0.2 M lithium sulphate. The same crystal form was also obtained by mixing the complex 1:1 with a solution of 0.1 M sodium cacodylate HCl pH 6.5, 2 M ammonium sulphate, 200 mM NaCl. The MERS1–RNA crystals took several days to 1 wk to grow. A MERS1–RNA crystal grown with the phosphate condition was ultimately used for data collection and structure determination. Data were collected for all crystals at the Advanced Light Source (ALS) beamline 8.3.1. The data were processed with MOSFLM and scaled with SCALA  using several previously solved structures with Nudix hydrolase folds were unsuccessful in solving the structure. Hence, selenomethionine (Semet) substituted protein was produced  to a final concentration of 1 mM. Crystals were grown using the hanging drop vapor diffusion method by mixing the complex 1:1 with a reservoir consisting of 19% PEG 3350 (w/v), 0.2 M magnesium formate. The crystals grew at room temperature and took from several weeks to months to grow. Apo MERS1 crystals were generated by mixing protein at 10 mg/mL with a crystallization reagent consisting of 0.1 M [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (HEPES) pH 7.5 and 1.6 M MgSOth SCALA . The MERproduced . The Semproduced . The selproduced . AUTOSOLproduced . There isolution . See TabRwork/Rfree values of 18.8%/20.9% and the model shows excellent geometry. MolProbity analysis places the structure in the top 100% of structures solved to a similar resolution , 5′-GGGAGGGGGUU-3′, 5′-GAAAGGGGGUU-3′, 5′-GAGGGGGGGUU-3′, 5′-GAGAAGGGGUU-3′, 5-GAGAGAGGGUU-3′, 5′-GAGAGGAGGUU-3′ and 5′-GAGAGGGAGUU-3′.FP experiments were performed using a PanVera Beacon 2000 FP system at 25°C . For theIn the FP peptide binding experiments, purified MERS1 was titrated into 0.990 mL buffer composed of 25 mM Tris HCl pH 7.5, 5% (v/v) glycerol and 150 mM NaCl and containing 1 nM  fluoresceintly labeled peptide. Data were fit using KaleidaGraph. Each binding curve is a representative curve from at least three technical replicates.For the SEC analyses of MERS1, the purified protein was concentrated to 4 mg/ml and loaded onto a Superdex 75 size exclusion column and eluted with a buffer containing 20 mM Tris HCl, pH 7.5, 150 mM NaCl, 5% (v/v) glycerol, and 1 mM DTT. The elution volume was compared to a series of protein standards to determine the molecular weights. The standards used for calculation of the standard curve are shown as blue diamonds and were cytochrome c oxidase (12.4 kDa), carbonic anhydrase (29 kDa) and albumin (66 kDa).Coordinates and structure factor amplitudes for the MERS1 GDP-bound, apo and RNA-bound structures have been deposited with the Protein Data Bank under accession codes 6P5R, 6NL1 and 6U9X, respectively.Supplemental material is available for this article.


Acropora hyacinthus, and Acropora spathulata, at two mid-shelf reefs on the Great Barrier Reef. By sampling over a 2-year time period, this study aimed to determine whether temporal variations reflect seasonal cycles. Community composition of both bacteria and Symbiodiniaceae was characterized through 16S rRNA gene and ITS2 rDNA metabarcoding. We observed significant variations in community composition of both bacteria and Symbiodiniaceae among time points for A. hyacinthus and A. spathulata. However, there was no evidence to suggest that temporal variations were cyclical in nature and represented seasonal variation. Clear evidence for differences in the microbial communities found between reefs suggests that reef location and coral species play a larger role than season in driving microbial community composition in corals. In order to identify the basis of temporal patterns in coral microbial community composition, future studies should employ longer time series of sampling at sufficient temporal resolution to identify the environmental correlates of microbiome variation.The coral microbiome is known to fluctuate in response to environmental variation and has been suggested to vary seasonally. However, most studies to date, particularly studies on bacterial communities, have examined temporal variation over a time frame of less than 1 year, which is insufficient to establish if microbiome variations are indeed seasonal in nature. The present study focused on expanding our understanding of long-term variability in microbial community composition using two common coral species,  Scleractinian corals are complex holobionts that host a high diversity and abundance of microbial symbionts that make up the coral microbiome , some ofThe coral microbiome is rarely static and its members can fluctuate as a result of changes in environmental conditions, or possibly due to host regulatory mechanisms , A. hyacinthus and A. spathulata, at two mid-shelf reefs in the central GBR, Rib reef , and Davies reef , were tagged and sampled over a 2-year time period. Both reef locations are similar distance from land, 75 and 79 km offshore, respectively. Sampling sites were back reef lagoon patch reef habitats and colonies were tagged at similar depths between 3–6 m. Sampling took place in February (end of summer) and October/November (end of winter) of both 2014 and 2015, as well as an additional time point in April 2015, making a total of five time points (2). Frozen samples were then freeze-dried and crushed using a hydraulic bench top laboratory press prior to DNA extraction. Average monthly temperature data over the duration of the study period from both Rib and Davies reef were obtained from publicly available data collected by the Australian Institute of Marine Science .Twelve colonies each of two species of coral, e points . At eachTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG AGGATT AGATACCCTGGTA-3′ and 1061R 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG CRRCACGAGCTGACGAC-3′ , 2 μL of each primer (2 μM stock), and 1 μL of DNA template, with an additional no template control to test for contamination. All 16S reactions were run on a Kyratec SC-200 thermal cycler (Kyratec Life Science) using the following protocol: initial denaturation at 95°C for 10 min, then 30 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 60 s and extension at 72°C for 60 s, followed by a final extension at 72°C for 7 min. The ITS2 PCR was also carried out in triplicate 10 μL reactions. Each reaction consisted of 5 μL of Qiagen Mulitplex MasterMix (Qiagen), 3 uL Milli Q, 0.5 μL of each primer (4 uM stock), and 1 μL of DNA template. All ITS2 reactions were run on a Kyratec SC-200 thermal cycler (Kyratec Life Science) using the following protocol: 95°C for 5 min, then 31 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, followed by a final extension at 72°C for 5 min. All PCR products were then examined using gel electrophoresis on a 2% TBE-agarose gel stained with Ethidium Bromide (EtBr). Some ITS2 products displayed double-banding, representing both the target and a mitochondrial band. The target bands were poked using the tip of a clean pipette, introduced to clean PCR master mix, and underwent a second PCR with the same specifications but only 12 cycles of denaturation, annealing, and extension. These products were again checked by gel electrophoresis to ensure no double-banding prior to sequencing. PCR clean-up, indexing and sequencing were carried out at the Ramaciotti Centre for Genomics at the University of New South Wales on a 2 bp × 300 bp Illumina MiSeq run. Data was returned as de-multiplexed paired-end sequences. Some coral samples had no amplification for either 16S or ITS2 and were thus left out of the downstream analyses  were identified and visualized as networks for each time point using Cytoscape v. 3.6.1  and for A. spathulata at Davies reef . Alpha diversity did not significantly vary over time for A. hyacinthus at Davies reef or A. spathulata at Rib reef  and for A. spathulata at both Rib  and Davies reefs , but not for A. hyacinthus at Davies reef.Alpha diversity significantly varied through time for Rib reef . ObserveEndozoicomonas, made up a higher percentage of the community in samples taken from Rib reef, where it made up 90.2 ± 2.2% and 70.3 ± 2.9% (mean ± SEM) for A. hyacinthus and A. spathulata, respectively. At Davies reef, Gammaproteobacteria accounted for 60 ± 3.5% for A. hyacinthus and 45.4 ± 3.9% for A. spathulata. An opposing pattern was observed for Betaproteobacteria; this class, which was dominated by the genus Burkholderia-Paraburkholderia, made up a higher percentage in samples taken at Davies reef as opposed to Rib reef. Betaproteobacteria at Davies reef accounted for 26.2 ± 2.9% and 34.3 ± 3.3% of the bacterial communities for A. hyacinthus and A. spathulata, respectively. At Rib reef, Betaproteobacteria made up only 4.54 ± 1.1% and 18.6 ± 2.02% of the communities for A. hyacinthus and A. spathulata, respectively.Bacterial communities of all samples were dominated by the classes Gammaproteobacteria, Betaproteobacteria, Bacilli, Alphaproteobacteria, and Deltaproteobacteria . GammaprA. hyacinthus at Rib reef , A. hyacinthus at Davies reef , A. spathulata at Rib reef  and A. spathulata at Davies reef . Pairwise PERMANOVA suggested no significant differences in the bacterial communities of A. hyacinthus among repeated sampling time points in February for Rib reef, but there were significant differences between the two February time points at Davies reef and between the October/November time points at both reefs  at Rib reef, but did show significant variations between repeated sampling points in February at Davies reef  of variation for both coral species and both reefs; 34 and 42% for A. hyacinthus at Rib and Davies reefs, respectively, and 14 and 39% for A. spathulata at Rib and Davies reefs, respectively.Beta-diversity significantly varied according to reef, species and time point . Due to th reefs . Pairwisies reef . For A. ints did . These direction . AccordiA. hyacinthus at Rib reef for February, and neither coral species at either reef showed any significant bacterial indicators for October/November time points. Two indicator taxa of the February time points were recovered for A. hyacinthus at Davies reef: one Endozoicomonas ASV and one Pseudoalteromonas. Three indicator taxa of the February time points were identified for A. spathulata at Davies reef, including two Endozoicomonas ASVs, one of which was the same ASV as found in A. spathulata at Davies reef, and one Vibrio. One Endozoicomonas ASV, which was different from the others, was found as the single indicator taxon of the February time points for A. spathulata at Rib reef  when all time points were considered together for each species at each reef  was not observed, small differences in the relative abundance or presence/absence of closely related bacterial strains were observed, which may have consequences for the functional potential of these bacterial communities (e.g., Endozoicomonas strains correlated both positively and negatively with each other. While the correlation between different Endozoicomonas strains could be a result of intragenomic variation (Endozoicomonas genomes have been found to host more than one copy of the 16S rRNA gene depending on species; Endozoicomonas sequence variants (The two coral species in the present study shared one es e.g., . Interesvariants . This hiA. hyacinthus and A. spathulata were complex and dynamic through time and variable according to reef and host species, yet not reflective of seasonality. This validates the findings of The microbiomes of PRJNA491379.The datasets generated for this study can be found in NCBI Sequence Read Archive, accession number: Anthozoans are not subject to ethics approval. Sampling for this study was carried out under the Great Barrier Reef Marine Park Authority and the Queensland Parks and Wildlife Service permit number G12/35236.1.HE, HS, GT, and MO developed the research question. VM and NC were responsible for the original sampling design and both funding for and provision of the coral samples. HE performed the genetic laboratory work, bioinformatics, and analyses with statistical assistance from HS. HE was responsible for the initial draft of the manuscript, with editorial support from all co-authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Schizosaccharomyces pombe. Combining the auxin-degron strain with the nda3 β-tubulin cold-sensitive (cs) mutant enabled us to inactivate condensin in mitotically arrested cells, without releasing the cells into anaphase. Transcriptional activation and termination were not affected by condensin's degron-mediated depletion, at heat-shock inducible genes or mitotically activated genes. On the other hand, condensin accumulation sites shifted approximately 500 bp downstream in the auxin-degron of 5′-3′ exoribonuclease Dhp1, in which transcripts became aberrantly elongated, suggesting that condensin accumulates at transcriptionally terminated DNA regions. Growth defects in mutant strains of 3′-processing ribonuclease and polyA cleavage factors were additive in condensin temperature-sensitive (ts) mutants. Considering condensin's in vitro activity to form double-stranded DNAs from unwound, single-stranded DNAs or DNA-RNA hybrids, condensin-mediated processing of mitotic transcripts at the 3′-end may be a prerequisite for faithful chromosome segregation.Condensin is an essential component of chromosome dynamics, including mitotic chromosome condensation and segregation, DNA repair, and development. Genome-wide localization of condensin is known to correlate with transcriptional activity. The functional relationship between condensin accumulation and transcription sites remains unclear, however. By constructing the auxin-inducible degron strain of condensin, herein we demonstrate that condensin does not affect transcription itself. Instead, RNA processing at transcriptional termination appears to define condensin accumulation sites during mitosis, in the fission yeast  Chromatin immunoprecipitation (ChIP) experiments showed that condensin occupancy at ribosomal DNA (rDNA) repeats is controlled by RNA polymerase I (RNAP I)-dependent transcription . T. Tcut3-4e (26°C) c. The seein Seb1 ,41 was a mutants d. Althou3.nda3 β-tubulin cs mutant. The aid-nda3 pair is a unique reagent, capable of inactivating the target protein during mitotic arrest, without releasing cells to anaphase, because the nda3 cs mutation produces an extremely high rate of synchronization (70–80%) in early mitosis (prophase-like stage) in S. pombe. Using the aid-nda3 strains, we next demonstrated that RNA processing at transcriptional termination defines condensin enrichment sites at mitotically activated genes, although condensin deficiency affects neither transcriptional activation nor termination. Our results reveal the selective accumulation of condensin at DNA regions in which the 3′-end of transcribed RNA should be processed. Condensin's action is presumably required especially under conditions in which processing of transcribed RNA and subsequent transcriptional termination are impacted, such as demonstrated in dhp1-aid strains. Additive growth defects between condensin ts and RNA processing mutants suggest that abnormally extended termination blocks mitotic progression in the absence of condensin activity. Although the mechanism by which improper RNA processing affects mitosis remains to be addressed, our finding supports the possibility that condensin acts on removal of mitotic transcripts from chromosome DNA for proper chromosome segregation , Swd2.2, and an ATP-dependent DNA/RNA helicase, Sen1, antagonize RNAP III-dependent transcription and condensin localization at RNAP III-transcribed genes [In ed genes ,43. Thisdhp1-aid cells, condensin may prefer specific nucleotide structures or proteins that accumulate at the shifted termination sites. Condensin binds to RNAP II-driven active genes in a transcription-dependent manner in fission yeast and human cells [S. pombe RNAP II enriches at 3′-ends of highly transcribed genes [S. pombe condensin SMC dimer and its holo-complex preferentially bind to ssDNA rather than dsDNA in vitro [et al. [in vivo. SMC dimer and holo-condensin promote the DNA renaturation reaction, which winds up complementary ssDNA to dsDNA in vitro [How does condensin accumulate at transcriptional termination sites? Considering the shift of condensin accumulation to 3′ downstream in an cells ,13. It ied genes ,45, sugged genes . Actuallin vitro . Sutani in vitro . In addiin vitro . TherefoS. pombe condensin enriches at DNA regions accumulating DNA-RNA hybrids, but they are unlikely to facilitate condensin recruitment [At RNAP III-transcribed gene loci, ruitment . It woulecm33+ [ecm33+ and gas1+ genes [The physiological significance of condensin accumulation at actively transcribed regions remains to be elucidated. By visualizing sister chromatid DNA separation, we recently showed that mutant condensin causes delayed segregation specifically at the mitotically transcribed, condensin-bound gene loci, ecm33+ . Contrarecm33+ ,47. Take1+ genes ,49. Cond4.4.1.S. pombe haploid wild-type strain 972 h− and its derivative mutant strains, including the temperature-sensitive (ts) cut14-208, cut14-y1 and cut3-477, and cold-sensitive (cs) dis3-54 and nda3-KM311, were used [rna14-393, dhp1-154, cft1-665, and seb1-41 ts mutants were isolated from a library of 1015 ts mutants [dhp1+, rna14+, and cut14+ genes were C-terminally tagged with HA and the target peptide of auxin, IAA17, and then integrated into strain FY21102, expressing the Skp1-AtTIR1-NLS-9myc fusion protein, as described previously [S. pombe were YPD and SPA sporulation medium [The ere used ,28,40. Sere used . The rna mutants . The dhpeviously . The FY24.2.nda3-KM311 mutant were performed as previously reported [6 cells ml−1 at 30°C (a permissive temperature), cultures were transferred to 20°C and incubated for 4 h to block spindle formation and to arrest cells at an early mitotic stage [Temperature-shift experiments using reported . When ceic stage . For aux4.3.ChIP was performed as previously described . Precipi4.4.S. pombe cells was extracted using the hot-phenol method [Total RNA from l method . One µg 4.5.S. pombe genomic DNA. Purified PCR fragments were labelled with Alkphos direct labelling  and detected with CDP-Star chemiluminescent detection reagents  using the LAS3000 imaging analyser (Fujifilm).A 1.2% agarose gel containing 6.3% formaldehyde in MOPS buffer was prepared and used for electrophoresis. 30 µg of total RNA was loaded into each lane. For probe preparation, PCR fragments of target genes were amplified from 4.6.Protein extracts were prepared by TCA precipitation . Cell cu4.7.DAPI-staining was carried out as previously described . All-in-