RNA-binding proteins typically change the fate of RNA, such as stability, translation or processing. Conversely, we recently uncovered that the small non-coding vault RNA 1-1 (vtRNA1-1) directly binds to the autophagic receptor p62/SQSTM1 and changes the protein's function. We refer to this process as ‘riboregulation'. Here, we discuss this newly uncovered vault RNA function against the background of three decades of vault RNA research. We highlight the vtRNA1-1-p62 interaction as an example of riboregulation of a key cellular process. VaultVaults are the largest ribonucleoprotein complexes known to date. They measure 400 × 400 × 700 Å and hence could enclose cellular structures bigger than ribosomes ,4. Vault2.VTRNA1 locus is situated between the ZMAT2 (zinc finger matrin-type 2) gene and the PCHD (protocadherin) cluster, while the VTRNA2 locus is found in close proximity, between TGFB1 (transforming growth factor beta 1) and SMAD5 (SMAD family member 5) on the same chromosome . ,11. 2,11nfection . FurtherVTRNA1-1 as well as other primate vault RNAs harbour a second copy of the box B motif and termination sequence downstream of the transcribed gene body that has been reported to negatively influence transcription depending on the upstream promoter sequences  ,21 11,21ppressor –46. By c fashion . The pro fashion . Converspression . Since v4.In addition to transcriptional regulation, post-transcriptional modifications and processing events contribute to the modulation of vault RNA abundance and function. Initial analyses revealed that the rat vault RNA is an uncapped RNA with a 5′ triphosphate (pppG) and an intact 3′ poly-uridine track ,17. It h5C; .–74.70–74b) ,82b) [75b) ,83,84. Sb) . The intb) [b) ,86. Strib) [b) , furtherb) [b) ,88. The b) [b) . It acco [b) [b) . In addi [b) [b) . Yet, thWe have recently shown that binding of vtRNA1-1 to p62 inhibits the receptor's oligomerization and engagement in autophagy. Mutagenesis of residues R139 and K141 to alanine in the ZZ domain of p62 reduced the interaction with vault RNA1-1. These residues are part of a positive surface patch in close proximity to the N-degron-binding site. Interestingly, we also observed decreased vtRNA1-1 binding to the p62 oligomerization mutant R21A/D68A/D73A (PB1 m) . These d8.Vault RNA levels decrease during starvation  and incrThe reduction of vtRNA1-1 expression during starvation is associated with a decrease in vtRNA1-1 binding to p62 . This deRecently, ZZ domain-specific ligands were shown to potently induce p62-dependent autophagy . VtRNA1-By contrast to starvation, viral infections induce vault RNA expression. This response was observed in human cell culture models that were infected with members of the γ-herpesviridae family, including Epstein–Barr virus (EBV/HHV4) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), alpha-herpesvirus  or paramyxovirus (Sendai virus (SeV) ,20,39,40in vitro and in vivo. Strikingly, Amort et al. [The transcriptional induction of vault RNAs upon infection has been associated with the expression of latent membrane protein 1 (LMP1) for EBV  and non-t et al.  showed tViruses are known to hijack key regulatory mechanisms of the cell to maximize viral replication while inhibiting cellular defence mechanisms. Upregulation of vtRNA1-1 levels could therefore serve to escape targeted viral degradation via autophagy and subsequent MHC class II antigen presentation . In addiFurthermore, the molecular pathways that mediate apoptosis resistance in this setting remain to be elucidated . Since pp62 levels decrease during autophagy due to phagolysosomal degradation  and incrThe intracellular levels of p62 can be used as a marker to assess the autophagic state of the cell . As mentIn contrast with the starvation-induced decrease in p62 levels, proteasome inhibition has been reported to stimulate the Nrf1-dependent transcription of p62 . This st9.The riboregulation of autophagy by a small non-coding RNA raises several additional perspectives, discussed below.9.1.Identification of p62 and vtRNA1-1 as a riboregulatory effector pair calls for the identification of those cellular and/or viral factors that determine the abundance of both components as well as their interaction. It will be of great interest to assess transcriptional, post-transcriptional and––in the case of p62––translational events that influence intracellular p62 and vault RNA1-1 levels. In addition, post-translational and epi-transcriptomic RNA modifications, respectively, could represent a fast way to control the RNA–protein interaction. While modifications within the binding interface could directly affect ribonucleoprotein complex formation, others that influence conformation or localization could indirectly contribute to the modulation of this interaction.5C has been functionally implicated in cell differentiation [Vault RNAs modifications have been recently described (see above), and mntiation ,55. Yet,ntiation . These intiation  within tntiation . Interesntiation . The ass9.2.It will be interesting to systematically determine the levels of p62 and vault RNA 1-1, and assess their interaction in cellular processes and stress conditions that are highly dependent on autophagy and its clearing function. These include differentiation and development , cell-deIn addition, p62 plays a pivotal role as a downstream effector for various other cellular pathways . Whi9.3.p62-dependent autophagy and aggregate clearance has been associated with neurodegenerative diseases, bone disorders and cancer ,99,108. Quality control and immunomodulatory functions of autophagy are considered to be tumour suppressive at early stages, while promotion of cellular growth and resistance to stress and drugs are believed to be tumour-promoting facets of autophagy in advanced tumours ,108–110.9.4.Previous work gave no positive indication of the involvement of MVP in the riboregulation of autophagy  or in ap9.5.α kinase PKR by viral RNAs in mammalian cells [Work on the vtRNA1-1-p62 interaction uncovered an example of riboregulation, a process in which a protein's function is controlled post-translationally by the direct binding of a regulatory RNA . Earlieran cells ,115 (revan cells ). With han cells  involvedan cells ,118.To systematically survey for the biological scope of riboregulation, it will be informative to determine the RNA-binding proteomes under various physiological and pathological conditions ,119–122.


Alu endo-siRNAs by cleaving long double-stranded regions of inverted Alu repeats. We identified AGO2-loaded Alu endo-siRNAs, which are highly expressed in commonly used cell lines. These Alu endo-siRNAs carrying both sense and antisense Alu sequences seem to target a set of genes containing a single Alu sequence, either antisense or sense, respectively, within their 3′UTR. In silico screening identified potential RNA silencing target genes for these Alu endo-siRNAs. We present results of a proof-of-concept experiment, in which sense Alu endo-siRNAs derived from AluSz and AluJr family elements target CUB Domain Containing Protein 1 mRNAs containing an antisense copy of AluJb in their 3′UTRs and consequently induce apoptosis in HeLa cells. Our results clearly indicate that Alu endo-siRNAs are functional also in somatic cells.In addition to adenosine-to-inosine RNA editing activities, ADAR1 has been shown to have various RNA editing-independent activities including modulation of RNAi efficacy. We previously reported that ADAR1 forms a heterodimer complex with DICER and facilitates processing of pre-miRNAs to mature miRNAs. In addition to miRNA synthesis, DICER is involved in processing of long dsRNAs into small RNAs (endo-siRNAs). Generation of retrotransposon-derived endo-siRNAs by DICER and their functions in regulation of transcripts in mouse oocytes has been previously reported. However, the synthesis and functions of endo-siRNAs in somatic cells remain largely unknown. Here, we report that ADAR1 together with DICER generates endogenous small RNAs,  Alu and SINE are the most frequent targets of ADAR1  specifically in double-stranded RNAs (dsRNAs) is catalyzed by adenosine deaminases acting on RNA (ADARs) . Three Aof ADAR1 . Studiesull mice . The cytin mRNAs . Interesu dsRNAs . Regardlu dsRNAs  and undeu dsRNAs . These Au dsRNAs .Vmax of the DICER pre-miRNA cleavage reaction and promotes loading of miRNA onto RISC, identifying a new role of ADAR1 in miRNA processing and RNAi mechanisms and revealing a stimulative interaction between RNA editing and RNAi  mRNAs containing an antisense copy of AluJb in their 3′UTRs  complexes as well as preparation of highly purified DICER alone and AluSx RNAs (302 nt in length). Differentially epitope tagged FLAG–DICER/HAT–ADAR1 and FLAG–DICER/HAT–TRBP complexes were purified by the baculovirus coexpression system and sequential epitope-based affinity chromatography purification as described previously  prior to establishment of our DICER protein purification protocol. Thus, the previously reported in vitro processing of Alu and 7SL RNAs into small fragmented RNAs by DICER could be due to contaminating RNases in the DICER preparation. Alternatively, the Alu or 7SL RNAs used for previous in vitro dicing assays might be contaminated by their antisense strand RNAs, which are often generated during in vitro transcription of sense strand RNAs. Synthesis of such antisense strand RNAs would result in the formation of completely complementary double-stranded Alu and 7SL RNAs: perfect substrates for DICER. We conclude that Alu RNAs transcribed by RNA polymerase III are not natural substrates of DICER, perhaps because of their rather short stem structures with many mismatched base pairs . Most interestingly, both ADAR1p110 and ADAR1p150 as DICER/ADAR1 complexes promoted DICER cleavage of Alu dsRNA to Alu siRNAs by approximately three- to fourfold , which is in the 3′UTR of NICN1 gene (NICN1 Alu dsRNA stem formed by sense and antisense of the same Alu element subfamily (AluSp) is much higher than that of the NFκB1 Alu dsRNA, and the loop size of the NICN1 Alu hairpin structure is much smaller than that of the NFκB1 Alu hairpin. In spite of these differences, we obtained similar results: The DICER/ADAR1 complex cleaved Alu dsRNA more efficiently and generated more Alu siRNAs than DICER alone . ADAR1 promotion of DICER activities to cleave pre-miRNAs to mature miRNAs has been reported to be independent of ADAR1 A-to-I editing activities , indicating that ADAR1 promotion of DICER cleavage of Alu dsRNA is also independent of the A-to-I editing activities of ADAR1.In addition to cleavage of pre-miRNAs to mature miRNAs, DICER also cleaves long dsRNAs into siRNAs . We nexting loop A. We firong loop A, which κB1 gene . We founfourfold B,C. In ccleavage B. We alsCN1 gene . Complemtivities . GeneratAlu siRNAs (∼21–24 nt) generated by cleavage of the NFκB1Alu dsRNA by the DICER/ADAR1p110 complex . Although we found that small RNAs were generated from both sense and antisense strands of the Alu hairpin stem region, their read numbers and patterns were not those expected from symmetrical processive cleavage of the completely matched dsRNA . We noticed no small RNA reads corresponding to certain regions containing internal loops and bulges. These results may indicate that DICER/ADAR1 complexes bind Alu dsRNA stems all along their length, not merely at the ends, and cleave them wherever bound, instead of acting processively in one direction.Alu dsRNA . In addition to adenosines of matched A:U base pairs, adenosines of mismatched A–C base pairs are known to be edited by ADAR1. ADAR1-mediated editing of adenosine residues of A:U matched base pairs results in I:U wobble base pairs, which decreases double-strandedness of Alu hairpin stems. On the other hand, editing of A–C mismatched base pairs results in I:C matched base pairs, which increases double-strandedness. Therefore, we next examined the effects of A-to-I editing on DICER cleavage of Alu dsRNAs. We first edited in vitro NFκB1 and NICN1 Alu dsRNA hairpins as much as possible using purified FLAG-ADAR1p110 proteins  prior to in vitro dicing assay. Interestingly, we found very little effects of A-to-I editing: Both unedited and edited Alu dsRNAs were cleaved by the DICER/ADAR1p110 complex to siRNAs equally well . Possibly due to the offsetting effects of A:U editing and A–C editing, A-to-I editing does not change significantly the overall double-strandedness of most Alu hairpins and consequently has very little effects on their DICER cleavage, in contrast to the reported antagonistic interaction of A-to-I editing and RNAi on long dsRNAs in C. elegans  has been also reported in HepG2.2.15 cells , although their expression levels were much lower than those of AGO2-bound miRNAs (only 0.09% of AGO2-bound miRNAs)  . We reexLa cells A, althou15 cells . We annofamilies B. In addfamilies . These Afamilies . We exam miRNAs) A. These Alu siRNAs, we examined Alu siRNA reads in ADAR1 knockdown HeLa cells (GSM105779)  and 30–32 (minor peak) nucleotides in length, are reduced significantly by ADAR1 depletion   in compaepletion A. As exp3T cells . These rAlu siRNAs identified in HEK293T cells . Whereas sense Alu siRNAs seemed to be generated from several separate regions of each Alu subfamily sequence , possibly indicating a preference of AGO2 binding to Alu siRNAs carrying a specific sequence.Annotation of AGO2-bound sense and antisense 3T cells A to knowu siRNAs B and Alusequence B, antiseAlu endo-siRNAs  indicate that they could certainly be engaged in RNA interference-mediated silencing of target genes, it is not known whether they are indeed functional in somatic cell lines. To obtain evidence for functionality of Alu endo-siRNAs in vivo, we chose four sense Alu siRNAs, S1 (AluSz), S2 (AluJb), S3 (AluJr), and S4 (AluSz) . Similarly, we also identified 165 genes containing a single copy of 3′UTR sense Alu sequences , which could be potential targets of antisense Alu siRNA, AS1–AS4 . Among the candidate genes containing a single copy of 3′UTR antisense Alu sequences, we further selected the genes known to regulate apoptosis, which can be easily determined as a phenotype of gene silencing . To this end, we picked CUB Domain Containing Protein 1 (CDCP1) containing a single antisense copy of AluJb subfamily sequence within its 3′UTR region . CDCP1, a transmembrane protein overexpressed in many types of cancers, suppresses apoptosis and thereby promotes their metastasis (Alu siRNAs (S1–S4) into HeLa cells. Their silencing effects were determined by western blotting analysis, which revealed that all four sense Alu siRNAs indeed suppressed expression of CDCP1. However, their silencing effects varied: S1, S3, and S4 were very effective, whereas S2 was least effective  B and looHeLa App , we firsR region  as a testastasis . We tranffective A. The diP1 mRNAs . Furtherequences . Their sequences B. As exp3 and S4 A as well3 and S4 B resulte3 and S4 A,B and a3 and S4 C. These Alu repeats within their 3′UTRs  lacking the amino-terminal DExD helicase domain, expressed only in oocytes, is highly active in processing endo-siRNAs from long dsRNAs  response via the MDA5-mediated dsRNA-sensing mechanism in contrast to in oocytes and embryonic stem cells where they enter into the RNA interference pathway and are processed to siRNAs by DICER. ADAR1 may contribute to control the potential of long dsRNAs for induction of the MDA5–MAVS–IFN pathway by two ways: introducing extensive A-to-I editing into dsRNAs and thereby suppress MDA5 binding in differentiated somatic cells or promoting the DICER activity and processing dsRNAs to siRNAs in oocytes and ES cells where the IFN pathway is absent. Interestingly, elimination of the IFN pathway components such as MAVS restored the dsRNA-mediated RNA interference pathway in mouse somatic cells, indicating that the dsRNA RNA interference pathway is present also in somatic cells but dominated by the IFN response pathway . In thisblockade . It is iSupplemental Table S1. Preparation of baculovirus constructs for HAT–ADAR1p110–WT, HA–ADAR1p110–E912A, and His–ADAR1p150–WT was described previously (AluSx sequence was synthesized by Gibson assembly and cloned into pBlueScript II KS (+). Oligonucleotides used for construction of various plasmids are listed in Supplemental Table S1. pBS-NFkB-dsAlu plasmid contains a 1457 bp fragment of an inverted Alu repeat of NFκB intron. The inverted Alu repeat located at chr1:11102837–11267747. The DNA fragment was PCR-amplified using human genomic DNA and PCR primers Bam-NFKBFW2 and Xho-NFKBDW2. Bam-NFKBFW2 contained a BamHI recognition site, and Xho-NFKBDW2 contained a XhoI recognition site. The PCR products were digested with BamHI and XhoI, then inserted into pBluescript II KS (−) vector  vector . pcDNA3.The recombinant proteins were purified with a TALON metal resin (Clontech) and/or an anti-FLAG M2 affinity or an anti-HA antibody bead (SIGMA) as described previously .32P-labeled Alu hairpin dsRNA substrates consisting of a partially double-stranded stem made from inverted sense and antisense Alu sequences and a loop and a single-stranded Alu RNA substrate were prepared by in vitro transcription as described previously , 0.05% NP40, 10% glycerol, 3U/µL RNasin plus inhibitor (Promega), 10 mM EGTA, 1.5 nM DICER, and 0.15 nM substrate RNA. Reaction mixtures were incubated at 37°C, and 7.5 µL aliquots were taken after 0, 5, 10, 30, and 60 min. The reactions were stopped by adding an equal volume of gel loading buffer . After heating at 80°C for 10 min, samples were analyzed by 10% Urea-PAGE and quantified using Molecular Dynamics and ImageJ (In vitro DICER cleavage assays were done in a 45 µL reaction mixture containing 20 mM PIPES (pH 6.2), 1.5 mM MgCld ImageJ .Secondary dsRNA structure was calculated using Mfold .We analyzed GSM1057798 , GSM1057799 , GSM1370372 , GSM1370373 , and GSM1334330 . We followed a multistep approach to align and annotate small RNA reads as follows: (i) removal of adaptor nucleotides; (ii) removal of siRNA sequences; (iii) alignment to ribosomal RNA sequences; (iv) alignment to transfer RNA sequences; (v) alignment to miRNA sequences; (vi) alignment to repetitive sequences; (vii) alignment to snoRNA sequences; (viii) alignment to RefSeq sequences; and (ix) alignment to human genome sequence. Alignment was performed using the Bowtie program allowing a maximum of two mismatches. One read reported one valid alignment. Unaligned reads were mapped to the next reference sequences. Bioinformatics analysis was performed on the Galaxy platform .https://www.arb-silva.de) (http://gtrnadb.ucsc.edu/index.html) (http://www.mirbase.org/ftp.shtml) (https://hgdownload.soe.ucsc.edu/downloads.html). The Rmsk file contains Alu subfamily sequences. (vii) Alignment to snoRNAs: The human snoRNA sequences were downloaded from snoRNABase (https://www-snorna.biotoul.fr/index.php) (https://hgdownload.soe.ucsc.edu/downloads.html). (ix) Alignment to human genome: The unaligned reads from the previous step were finally aligned to the hg38 human genome.More specifically: (i) Removal of adaptor: The 3′ adaptor sequences  were first removed from the sequences. Trimmed reads of length 19 to 35 nt were subjected to further analysis. (ii) Removal of exogenous siRNA sequences: The reads containing the control siRNA and ADAR1 siRNA sequences used were removed. Reads with 5′-CGTACGCGGAATACTTCGAAG-3′, 5′-TCGAAGTATTCCGCGTACGAT-3′, 5′-CCGCCATCATTATGAAAAAAG-3′ and 5′-TTTTTCATAATGATGGCGGAT-3′ sequences were removed. (iii) Alignment to ribosomal RNAs: The human ribosomal RNA sequences were downloaded from Silva (ilva.de) . (iv) Alex.html) . (v) Alip.shtml) . (vi) Aldex.php) . (viii) The alignment data were visualized by the Integrative Genomics Viewer.Alu sequences and genes with exactly one Alu repeat in the forward orientation (sense) to the gene or exactly one Alu in reverse orientation (antisense) to the gene were reported. Highly expressed genes in HeLa cells (CCL2) were picked using HeLa App (proteomics: >log10 4.5). The presence of articles related to apoptosis was examined using a keyword search of the abstract in PubMed.Coding genes with known 3′UTR regions were tested for 2. HeLa cells were free from mycoplasma contamination. Small-RNA transfection experiments were performed with Lipofectamine RNAiMax (Life Technologies) with a 2 nM final RNA concentration. All small-RNAs used in this study are listed in Supplemental Table S1. Silencer Select siRNAs for human CDCP1(s35060 and s35061) and Negative Control siRNA (s4390844) were purchased from Life Technologies .HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (GEMINI) and penicillin/streptomycin at 37°C in 5% COSupplemental Table S2) overnight at 4°C. After incubation with secondary antibodies, membranes were developed with ECL .Cell lysates were prepared in Laemmli buffer (Boston BioProducts) containing benzonase nuclease (Sigma), Complete EDTA-free protease-inhibitor cocktail (Roche), and PhosStop phosphatase-inhibitor cocktail (Roche), and fractionated by 4%–15% SDS–PAGE. Proteins were transferred to Immobilon-P nylon membranes (Millipore). Membranes were blocked with 1% Blocker BSA (Life Technologies) and incubated with primary antibodies , 0.05% NP40, 10% glycerol, 3 U/mL RNasin plus (Promega), 10 mM EGTA, 1.5 nM DICER/ADAR1p110-WT complexes, and 3 nM NFκB1 dsAlu RNA. DICER cleavage products were purified from 10% acrylamide denaturing gel and ligated with 5′ and 3′ adaptors. 3′ cloning adaptors were ligated to the cleavage products using T4 RNA ligase 2-truncated K227Q in a reaction buffer containing NEB ligation buffer, 15% PEG8000, and 1U/µL RNasin plus inhibitor. After denaturing gel purification, 5′ cloning adaptors were ligated to the purified RNAs using T4 RNA ligase1 in a reaction buffer containing NEB ligation buffer, 20% PEG8000, 1U/µL RNasin plus inhibitor, 20 µM ATP, and 10% DMSO. RNA ligation reactions were incubated at 22°C for 120 min. After denaturing gel purification, the ligated RNAs were reverse-transcribed using the RT-primer and SuperScript III (Life Technologies). The reverse-transcribed products were amplified using first and second PCR primers and AccuPrime high fidelity Taq DNA polymerase (Life Technologies). After acrylamide gel purification, PCR products were sequenced by Ion PGM  (Life Technologies). Bioinformatics analysis: The 5′ and 3′ adaptor sequences  were removed from the sequences. Trimmed reads were then mapped to NFκB1 intronic inverted Alu sequence. Alignment was performed using the Bowtie program without any mismatch. Bioinformatics analysis was performed on the Galaxy platform (In vitro DICER cleavage assays for sequencing were done in a 100 µL reaction mixture containing 20 mM PIPES (pH 6.2), 1.5 mM MgClplatform .CDCP1 mRNA coding region or Alu sense siRNAs (S3–S4)  were used for knockdown of the CDCP1 gene. Staurosporine  treatment at 2 µM for 2 h was used as positive control. DNA was counterstained with Hoechst 33342 (Life Technologies). Apoptosis levels were also determined by western blotting analysis using cleaved PARP (Asp214) antibody .Apoptotic cells were analyzed by fluorescence microscopy using CellEvent Caspase-3/7 Green apoptosis-detection system (Life Technologies), which utilizes a fluorogenic substrate for activated Caspase-3 and -7. Synthetic siRNAs corresponding to the human t-tests were conducted, and the minimum level for significance was P < 0.05.Image quantification was performed with ImageJ software. Data are presented as means ± SD. Two-tailed Dicing product sequencing data has been deposited in the Sequence Read Archive under series accession number SRR10824153.Supplemental material is available for this article.


Type VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified Cas13bt3  as a miniature Cas13 enzyme, which can be used for knockdown and editing of target transcripts in mammalian cells. However, the action mechanism of the compact Cas13bt3 remains unknown. Here, we report the structures of the Cas13bt3-guide RNA complex and the Cas13bt3-guide RNA-target RNA complex. The structures revealed how Cas13bt3 recognizes the guide RNA and its target RNA and provided insights into the activation mechanism of Cas13bt3, which is distinct from those of the other Cas13a/d enzymes. Furthermore, we rationally engineered enhanced Cas13bt3 variants and ultracompact RNA base editors. Overall, this study improves our mechanistic understanding of the CRISPR-Cas13 enzymes and paves the way for the development of efficient Cas13-mediated transcriptome modulation technologies.  The class 2 systems are further divided into three types, II, V, and VI, in which Cas9, Cas12, and Cas13 function as effector proteins, respectively. Cas9 associates with a crRNA and a tracrRNA and cleaves double-stranded DNA (dsDNA) targets, using the RuvC and HNH nuclease domains (CRISPR-Cas systems in prokaryotes provide adaptive immunity against foreign nucleic acids and are divided into two classes (classes 1 and 2) . In the  domains . Althoug domains . Since s domains .cis and bystander ssRNAs in trans   nuclease domains . Cas13 aleavage) . Cas13 hleavage) . The Casleavage)  and sharleavage) .Cas13a has been harnessed for RNA knockdown and detection , 2018. ICas13bt3  was recently identified as a minimal Cas13 protein (775 residues) and used for RNA knockdown in mammalian cells . FurtherLeptotrichia shahii Cas13a -crRNA-target RNA ternary complex revealed that, upon target RNA binding, the HEPN domains undergo structural changes and adopt the cleavage-competent active conformation, thereby providing important information about the activation mechanisms of the Cas13a enzymes (Eubacterium siraeum Cas13d (EsiCas13d) in distinct functional states illuminated the Cas13d-mediated RNA cleavage mechanism, which is similar to that of Cas13a . In addition, the crystal structures of Cas13b-crRNA binary complexes, such as Bergeyella zoohelcum Cas13b (BzoCas13b) (Prevotella buccae Cas13b (PbuCas13b)   and PrevuCas13b) , revealeHere, we report the crystal structure of Cas13bt3 in complex with a crRNA and the cryo-EM structure of Cas13bt3 in complex with a crRNA and its complementary target RNA. These structures, along with our mutational experiments and molecular dynamics (MD) simulations, provide mechanistic insights into crRNA recognition and target RNA cleavage by Cas13bt3, which are distinct from those of the previously described Cas13a/d enzymes. Furthermore, we rationally engineered Cas13bt3 variants with improved efficiency and specificity. Overall, these findings enhance our understanding of the diversity of the type VI CRISPR-Cas13 enzymes and establish a framework for Cas13-based therapeutic RNA targeting and editing.To understand how Cas13bt3 specifically assembles with its cognate crRNA, we solved the crystal structure of the catalytically inactive dCas13bt3 (R84A/H89A/R739A/H744A) in complex with a crRNA consisting of 36-nucleotide DR and 5-nucleotide spacer sequences , at 1.9-Å resolution ; Table 1The HEPN1 domain (residues 1–184) comprises nine α helices and two β strands, while the HEPN2 domain (residues 616–775) contains seven α helices and four β strands . The HEPThe crRNA consists of the 5-nucleotide spacer (guide) segment and the 36-nucleotide DR region .The DR rThe crRNA DR is recognized by the Helical-1, Lid, and Helical-2 domains  and S3. in vitro RNA cleavage assays demonstrated that double mismatches at the central region (mm3), but not the 5′ and 3′ regions (mm1 and mm4), of the spacer reduced the Cas13bt3-catalyzed RNA cleavage  and its complementary target RNA (25 nucleotides), at an overall resolution of 3.4 Å ; Table 2in vitro. The K645A mutation reduced the RNA cleavage  values for the Cα atoms in the binary and ternary complexes during the 200-ns simulations. In two independent simulations, the NUC lobe exhibited higher RMSF values in the ternary complex as compared with the binary complex, whereas the REC lobe displayed comparable RMSF values in the two complexes , suggestThese structural and functional analyses, together with our MD simulations, provided mechanistic insights into the Cas13bt3-mediated RNA-guided RNA cleavage. In the Cas13bt3-crRNA binary complex, the HEPN active site is covered by the ASL and thus inaccessible by ssRNA substrates. The Cas13bt3-crRNA complex recognizes a complementary ssRNA to form the crRNA-target RNA duplex, which facilitates the rearrangement of the HEPN domains. In the Cas13bt3-crRNA-target RNA ternary complex, the HEPN domains move away from the REC lobe, and thus, the ASL becomes more flexible and dissociates from the HEPN active site, thereby allowing the access of ssRNA substrates to the catalytic residues. The ASLs in the PbuCas13b and BzoCas13b binary complexes cover their HEPN active sites, as in the Cas13bt3 binary complex , suggestTo improve the utility of Cas13bt3 in RNA targeting and editing, we sought to engineer a Cas13bt3 variant with better RNA-binding ability. We selected 17 residues and designed 28 Cas13bt3 mutants with amino acid substitutions that may provide new interactions within the Cas13bt3-crRNA-target RNA complex . Among tTo evaluate the RNA-targeting ability of enCas13bt3, we measured RNA knockdown by the wild-type Cas13bt3 and en-Cas13bt3 in HEK293FT cells, using two different crRNAs (crRNAs 1 and 2) targeting a luciferase reporter gene. en-Cas13bt3 exhibited higher knockdown efficiency, as compared with the wild-type Cas13bt3 . To examA recent study reported that a Cas13bt3 variant (referred to as miniCas13X), lacking the HEPN1 (residues 1–180) and HEPN2 domains (residues 626–775), functions as a compact RNA-targeting platform and can be used for RNA editing , consistIn this study, we determined the crystal structure of the Cas13bt3-crRNA binary complex, revealing how Cas13bt3 recognizes its cognate crRNA to form the compact effector complex. A structural comparison of Cas13bt3 with other Cas13b enzymes highlighted their architectural conservation and divergence, providing a structural explanation for the miniaturization of the Cas13bt3 scaffold. We also determined the cryo-EM structure of the Cas13bt3-crRNA-target RNA ternary complex, which represents the first ternary complex structure of the Cas13b family enzymes. The present Cas13bt3 structures, together with our functional and computational analyses, have provided insights into the Cas13b-mediated RNA cleavage mechanisms.A structural comparison of Cas13bt3 with Cas13a  and Cas1We rationally engineered the enCas13bt3 variant, which contains the E172R and E297F substitutions and exhibits enhanced RNA-targeting activity. The Cas13bt3 structure suggested that Arg172 (E172R) reinforces the interaction with the crRNA backbone phosphate, similar to substitutions that enhance protein-DNA interactions in the Cas9 variants . These rIn conclusion, these Cas13bt3 structures provided mechanistic insights into the miniaturization of Cas13bt3 and the RNA cleavage by the Cas13b family enzymes. Furthermore, we rationally engineered Cas13bt3 variants, such as en-Cas13bt3 and miniCas13bt3-REPAIR. Our findings expand the understanding of diverse type VI CRISPR-Cas13 enzymes and will facilitate the development of efficient RNA-editing technologies.Our structural, functional, and computational data suggested that the activation mechanism of Cas13bt3 is distinct from those of the Cas13a/Cas13d enzymes. Nonetheless, the NUC lobe in the ternary complex structure was not well resolved in the density map, probably due to its flexibility. Therefore, it will be important to investigate the functional role of the ASL for the Cas13bt3 activation in the future. In addition, structural analyses of other Cas13b enzymes bound to target RNAs will be required to understand the activation mechanism of Cas13b family enzymes.nureki@bs.s.u-tokyo.ac.jp).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Osamu Nureki (Escherichia coli cells were cultured at 37°C in LB medium (containing 20 mg/l kanamycin) for plasmid and protein preparation. HEK293FT cells were grown at 37°C in Dulbecco’s Modified Eagle Medium with high glucose, sodium pyruvate, and GlutaMAX (Thermo Fisher Scientific), supplemented with 1 × penicillin–streptomycin (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), and 10% fetal bovine serum (VWR Seradigm).The atomic models have been deposited in the Protein Data Bank under the accession codes 6VTI (binary complex) and 6VTN (ternary complex). The cryo-EM density map has been deposited in the Electron Microscopy Data Bank under the accession code EMD-32118 (ternary complex). Raw movies of the cryo-EM dataset have been deposited in the Electron Microscopy Public Image Archive under the accession code EMPIAR-11110. The data of unprocessed image files have been deposited in the Mendeley Data repository (doi: 10.17632/nst3mp7h2t.1).This paper does not report original code.Any additional information required to reanalyze the data reported in this paper is available from the Lead Contact upon request.All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.6-tagged Cas13bt3 protein was expressed in E. coli Rosetta2 (DE3). The E. coli cells were cultured at 37°C until the OD600 reached 0.8, and protein expression was then induced by the addition of 0.1 mM isopropyl β-D-thiogalactopyranoside . The E. coli cells were further cultured at 20°C overnight, harvested by centrifugation, resuspended in buffer Å , and then lysed by sonication. The lysates were centrifuged, and the supernatant was mixed with 3 mL Ni-NTA Superflow resin (QIAGEN). The mixture was loaded into a Poly-Prep Column (Bio-Rad), and the protein was eluted with buffer B . The protein was incubated with TEV protease overnight, and then loaded onto a 5 mL HiTrap SP column , equilibrated with buffer C . The protein was eluted with a linear gradient of 0.3–2 M NaCl, and then further purified on a Hiload Superdex 200 column , equilibrated with buffer D . The purified protein was stored at –80°C until use. For structural studies, the crRNA and the target RNA were purchased from Ajinomoto Bio-Pharma. For in vitro RNA cleavage experiments, the crRNAs and the target RNA were transcribed in vitro with T7 RNA polymerase, and purified by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis (PAGE)  was synthesized by GenScript and cloned into the modified pE-SUMO vector (LifeSensors). Mutations were introduced by a PCR-based method, and sequences were confirmed by DNA sequencing . The N-ts (PAGE) .2, and 1 mM DTT). The purified complex was crystallized at 20°C by the hanging-drop vapor diffusion method. Crystals were obtained by mixing 1 μL of complex solution (A260 nm = 15) and 1 μL of reservoir solution . The SeMet-labeled protein was crystallized under conditions similar to those for the native protein. X-ray diffraction data of the SeMet-labeled protein were collected at 100 K using an EIGER X16M detector (Dectris) and a wavelength of 0.9780 Å on beamline BL41XU at SPring-8 . The crystals were cryoprotected in reservoir solution supplemented with 25% ethylene glycol. X-ray diffraction data were processed using KAMO (The Cas13bt3-crRNA binary complex was reconstituted by mixing the purified dCas13bt3 (R84A/H89A/R739A/H744A) and the 41-nucleotide crRNA at a molar ratio of 1:1.2. The binary complex was purified on a Superdex 200 Increase 10/300 column , equilibrated with buffer E . The purified complex solution (A260 nm = 6) was applied to freshly glow-discharged Au/Rh 300 mesh R1.2/1.3 grids (Quantifoil) on both sides, using a Vitrobot Mark IV (FEI) at 4°C, with a waiting time of 10 s and a blotting time of 4 s under 100% humidity conditions. The grids were plunge-frozen in liquid ethane cooled at liquid nitrogen temperature.The Cas13bt3=crRNA-target RNA ternary complex was reconstituted by mixing the purified dCas13bt3 (R84A/H89A/R739A/H744A), the 61-nucleotide crRNA, and the 25-nucleotide target RNA at a molar ratio of 1:1.2:1.4. The ternary complex was purified on a Superdex 200 Increase 10/300 column, equilibrated with buffer F , running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode . Movies were recorded at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.83 Å at the electron exposure of 13 esoftware , with a software  implemensoftware , and thesoftware .overall), according to the Fourier shell correlation (FSC) = 0.143 criterion (NUC), and this map was used for modeling. The local resolution was estimated by RELION-3.1.Data were processed using RELION-3.1. From the 2,727 motion-corrected and dose-weighted micrographs, 2,380,559 particles were initially picked and extracted at a pixel size of 3.26 Å. These particles were subjected to several rounds of 2D and 3D classifications. The 582,042 best selected particles were then re-extracted at a pixel size of 1.22 Å and subjected to 3D refinement. The resulting 3D model and particle set were subjected to per-particle defocus refinement, beam-tilt refinement, and Bayesian polishing . The 3D riterion . Whereashttp://www.pymol.org) and CueMol (http://www.cuemol.org).The model of the binary complex was automatically built using Buccaneer  and NautCas13bt and related Cas13b sequences were retrieved from the NCBI, using a psi-blast search with Cas13bt1 as the query sequence on the HH-suite NR90 database with an E-value reporting and gathering threshold of 1e-3 . Sequencin vitro, using a 5′-Cy5-labeled target RNA containing a 30-nucleotide spacer-complementary sequence  and Proteinase K (40 ng). The reaction products were fractionated by 10% denaturing (7 M urea) PAGE, and then visualized using an Amersham Imager 600 . In vitro RNA cleavage experiments were performed at least three times.The RNA cleavage activity of Cas13bt3 was measured sequence . The Casin vitro, using the purified Cas13bt3 and its pre-crRNA containing two spacer-DR units. The Cas13bt3 protein  was mixed with the pre-crRNA , and then incubated at 37°C for 2 h in 20 μL processing buffer, containing 20 mM HEPES-NaOH, pH 7.5, 50 mM NaCl, and 0.5 mM MgCl2. The reaction was stopped by the addition of quench buffer. The reaction products were then fractionated by 10% denaturing (7 M urea) PAGE. The gels were stained with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific). The purified PbuCas13b and its pre-crRNA were used as the positive control. In vitro processing experiments were performed at least three times.The pre-crRNA processing was examined 3, and contained 197,387 and 196,537 atoms in the binary and ternary complexes, respectively. The molecular topologies and parameters from the Charmm36 force field , and missing hydrogen atoms were built using VMD . The netce field  were usece field . The simn piston . The lonn piston . The simn piston , Seaborn4 cells/well 16–20 h prior to transfection, to ensure 90% confluency at the time of transfection. For each well on the plate, transfection plasmids were combined with OptiMEM I Reduced Serum Medium (Thermo Fisher Scientific) to a total of 25μL. Separately, 23 μL of OptiMEM was combined with 2 μL of Lipofectamine 2000. The plasmid and Lipofectamine solutions were then combined and pipetted onto cells.Mammalian cell culture experiments were performed with the HEK293FT line (American Type Culture Collection (ATCC)) grown in Dulbecco’s Modified Eagle Medium with high glucose, sodium pyruvate, and GlutaMAX (Thermo Fisher Scientific), supplemented with 1 × penicillin–streptomycin (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), and 10% fetal bovine serum (VWR Seradigm). All cells were maintained at confluency below 80%. All transfections were performed with Lipofectamine 2000 (Thermo Fisher Scientific) in 96-well plates unless otherwise noted. Cells were plated at 2×10Gaussia/Cypridina(W85X) luciferase reporter plasmid (Cypridina and Gaussia luciferase activities in the media were measured using Gaussia and Cypridina Luciferase Assay Kits (Targeting Systems) with the injection protocol on a Biotek Synergy Neo 2 (Agilent) imaging reader. Each experimental luciferase measurement was normalized to the appropriate control luciferase measurement .HEK293FT cells were transfected as described with 25 ng of a plasmid encoding the Cas13bt3 variant expressed from a CMV promoter, 300 ng of a plasmid encoding a crRNA expressed from a human U6 promoter, and 45 ng of a dual  plasmid  (Table S plasmid . After 4Gaussia/ Cypridina(W85X) luciferase reporter plasmid (Cypridian luciferase (5′-TTTGCATTCATCTGGTACTTCTAGGGTGTC-3′). cDNA was used as input for the preparation of next-generation sequencing libraries with NEBNext High-Fidelity 2 × PCR Master Mix (NEB), and amplicons were then sequenced on an Illumina MiSeq. Editing was quantified by counting the number of reads at which the expected edited position in the amplicon was identified as G and dividing by the total number of reads in the sample, using Python. Unless otherwise noted, all reported data are the average of four biological replicates. To measure the restoration of the Cypridina luciferase (W85X) activity, the culture media were aspirated from the same cell samples and the Cypridina and Gaussia luciferase activities in the media were measured as described above.HEK293FT cells were transfected as described with 25 ng of a plasmid encoding a dCas13bt3-ADAR2dd(E488Q) fusion expressed from a CMV promoter, 300 ng of a plasmid encoding a crRNA expressed from a human U6 promoter, and 45 ng of a dual  plasmid  (Table S plasmid . After 4 plasmid , using aIn vitro experiments were performed at least three times.Article plus Supplementary InformationFigures S1-S7 & Table S1


Alu retroelements is a primate-specific mechanism mediated by adenosine deaminases acting on RNA (ADARs) that diversifies transcriptome by changing selected nucleotides in RNA molecules. We tested the hypothesis that A-to-I RNA editing is altered in rheumatoid arthritis (RA).Adenosine-to-inosine (A-to-I) RNA editing of AluSx+ Sanger-sequencing of cathepsin S, an extracellular matrix degradation enzyme involved in antigen presentation.Synovium expression analysis of ADAR1 was investigated in 152 RA patients and 50 controls. Peripheral blood mononuclear cells derived from 14 healthy subjects and 19 patients with active RA at baseline and after 12-week treatment were examined for ADAR1p150 and ADAR1p110 isoform expression by RT-qPCR. RNA editing activity was analysed by AluSx+ decreased after treatment only in those patients with good clinical response. Upregulation of the expression and/or activity of the RNA editing machinery were associated with a higher expression of edited Alu-enriched genes including cathepsin S and TNF receptor-associated factors 1,2,3 and 5.ADAR1 was significantly over-expressed in RA synovium regardless of disease duration. Similarly, ADAR1p150 isoform expression was significantly increased in the blood of active RA patients. Individual nucleotide analysis revealed that A-to-I RNA editing rate was also significantly increased in RA patients. Both baseline ADAR1p150 expression and individual adenosine RNA editing rate of cathepsin S A previously unrecognized regulation and role of ADAR1p150-mediated A-to-I RNA editing in post-transcriptional control in RA underpins therapeutic response and fuels inflammatory gene expression, thus representing an interesting therapeutic target. •The RNA editing enzyme ADAR1 is increased in rheumatoid arthritis.•Alu A-to-I RNA editing is increased in active RA and decreases after treatment only in responding patients.ADAR1-induced •Association of increased A-to-I RNA editing with pro-inflammatory gene expression suggests a role in chronic inflammation.•Alu RNA editing may comprise an interesting therapeutic target in RA.Interrupting the pro-inflammatory ADAR1p150-induced  HoweverAlu elements, which have the ability to form double-stranded RNA regions, a pre-requisite for RNA editing  and the 10.13039/100004807DFG (SFB834 project number 75732319) to K. Stellos; ELKE grants from the 10.13039/501100005187National and Kapodistrian University of Athens, Greece to P.P. Sfikakis [grant number: 0974]; AIRC IG n. 22080 to A.Gallo; and a scholarship for doctoral studies to N.I.V by A. Onassis Public Benefit Foundation.This work was supported by the All participants gave written informed consent, which had been previously approved by the Ethics Committee of Laiko Hospital, Athens, Greece (Protocol Nr.:1368/17-11-2016), as well as by the Hellenic Data Protection Authority (Protocol Nr.:ΓΝ/ΕΞ/7901-2/22-12-2016).RNA-sequencing dataset GSE89408 is publicly available at GEO database (NCBI). Raw data of the qPCR experiments and RNA editing analysis are available by the corresponding author upon reasonable request.We did not involve patients or the public in our work.


Streptococcus suis serotype pairs 2 and 1/2 as well as 1 and 14, respectively, based on a single‐nucleotide polymorphism within capsular polysaccharide synthesis gene cluster K. This assay is easy to implement and identifies potential zoonotic serotypes.This rapid high resolution melting (HRM) assay allows distinguishing between  Streptococcus suis serotype pair 2 and 1/2 or 1 and 14, respectively. The assay is based on a single‐nucleotide polymorphism within the capsular polysaccharide synthesis gene cluster K and enables a rapid identification of potential zoonotic serotypes.The designed high resolution melting assay allows to correctly differentiate  Strains were identified by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry . Genomic DNA was extracted using a standard heat lysis protocol  at base pair position 483 , representing the repeatability of the developed HRM assay, all isolates were tested. The variability assays were performed in triplicates in three independent runs at three different days.The HRM assay was evaluated, and its specificity was determined. To examine the intra‐ and interassay variability of the melting temperatures (3S. suis strains into two clusters grouping serotype 2 with 14 and serotype 1/2 with 1 (Figure S. suis isolate. Based on our results, the target was 100% specific for the four serotypes tested as none of the Streptococcus spp. or S. suis with different serotypes (Table Tm were between 0.008% and 0.03% and interassay CVs, respectively, between 0.04% and 0.07% illustrating a highly reproducible and stable assay. Serotypes 1 and 1/2 yielded a mT of 75.9 ± 0.1°C, whereas serotypes 2 and 14 yielded a Tm of 76.2 ± 0.1°C, respectively (Figure The HRM assay clearly divided the 17 1 Figure  into ser4S. suis strains and helps strain tracking in case of disease outbreaks. In addition, this assay provides an easily applicable diagnostic tool for high‐throughput screening and rapid identification of S. suis serotypes 2 and 14 relevant for human infection without the need of a WGS approach. Compared with Sanger sequencing and WGS, a HRM assay is not depended on manual inspection of the sequencing data and results are obtained straightforward in less than 1.5 hr. Reagent costs are low with about 1 CHF per sample in comparison to Sanger sequencing, which was around ten times as expensive with an estimated prize of 10 CHF per sample. The cost of WGS sequencing of approximately 200 CHF per sample is not competitive if the focus of investigation is set at serotype differentiation or at screening of potentially zoonotic isolates. Even though, the local reagents and labor cost may be variable, HRM is by far the most cost efficient and easily applicable assay delivering accurate results in a short turnaround time. Thus, a possible surveillance system contributing to the development of proficient public health policies can be established.In this report, a novel HRM assay based on SNP detection in PCR products is described. HRM analysis is a rapid and low cost genotyping method simply convertible in laboratories as a singleplex method with a low risk of contamination (Reed, Kent, & Wittwer, 5cpsK. This assay allows a rapid separation of these S. suis serotypes in routine diagnostic laboratories using molecular based serotyping in order to assess a zoonotic risk of emerging strains.To conclude, we have developed a specific HRM assay distinguishing between serotype pairs 2 and 1/2 and 1 and 14, respectively, based on a SNP within None declared.Simone Scherrer: Conceptualization‐Equal, Investigation‐Equal, Validation‐Lead, Writing‐original draft‐Lead; Fenja Rademacher: Investigation‐Supporting; Nathalie Spoerry Serrano: Investigation‐Supporting; Jacques Schrenzel: Resources‐Equal, Writing‐review and editing‐Supporting; Marcelo Gottschalk: Resources‐Equal, Writing‐review and editing‐Supporting; Roger Stephan: Conceptualization‐Equal, Supervision‐Lead, Writing‐review and editing‐Lead; Patricia Landolt: Conceptualization‐Equal, Investigation‐Equal, Validation‐Supporting, Writing‐review and editing‐Supporting.None required.


Spatiotemporal translational regulation plays a key role in determining cell fate and function. Specifically, in neurons, local translation in dendrites is essential for synaptic plasticity and long-term memory formation. To achieve local translation, RNA-binding proteins in RNA granules regulate target mRNA stability, localization, and translation. To date, mRNAs localized to dendrites have been identified by comprehensive analyses. In addition, mRNAs associated with and regulated by RNA-binding proteins have been identified using various methods in many studies. However, the results obtained from these numerous studies have not been compiled together. In this review, we have catalogued mRNAs that are localized to dendrites and are associated with and regulated by the RNA-binding proteins fragile X mental retardation protein (FMRP), RNA granule protein 105 , Ras-GAP SH3 domain binding protein (G3BP), cytoplasmic polyadenylation element binding protein 1 (CPEB1), and staufen double-stranded RNA binding proteins 1 and 2 (Stau1 and Stau2) in RNA granules. This review provides comprehensive information on dendritic mRNAs, the neuronal functions of mRNA-encoded proteins, the association of dendritic mRNAs with RNA-binding proteins in RNA granules, and the effects of RNA-binding proteins on mRNA regulation. These findings provide insights into the mechanistic basis of protein-synthesis-dependent synaptic plasticity and memory formation and contribute to future efforts to understand the physiological implications of local regulation of dendritic mRNAs in neurons. Spatiotemporal translational regulation is key to increasing the concentration of specific proteins to exert their functions at specific timings and locations in cells. In neurons, mRNAs are translated not only in the cell soma but also in axons and dendrites ,2. LocalIn 1996, Kang and Schuman revealed that long-term potentiation (LTP) occurs in the stratum radiatum (SR), which is a dendrite-enriched region in hippocampal CA1, even after the SR was isolated from the cell-soma-enriched stratum pyramidale (SP). LTP in the isolated SR was lost after inhibition of protein synthesis . Their fThis review catalogues and selects mRNAs that are localized to dendrites and regulated by RNA-binding proteins in RNA granules. Here, we focus on dendritic mRNAs that have been identified in common in several studies that identified dendritic mRNAs based on various criteria using comprehensive RNA sequencing (RNA-seq) of the SR from rodent hippocampal CA1. These mRNAs are further classified into groups according to the functions of the mRNA-encoded proteins. In addition, we focus on the major RNA-binding proteins in RNA granules and list their target mRNAs and effects on mRNAs. Furthermore, we compared our selected set of dendritic mRNAs with the RNA-binding protein target mRNAs and discuss the contribution of RNA-binding proteins to mRNA regulation, such as mRNA expression, stability, localization, and translation, for the regulation of local translation in dendrites.Camk2a, Map2, Arc, and Insp3r1 mRNAs were intensively studied . Th. Th15]. Nakayama et al. identified mRNAs more enriched in the SR layer compared with the SP layer in the hippocampus of adult mice  16]. Th. Th16]. Ainsley et al. identified ribosome-bound mRNAs in the hippocampus of mice that had received a novel experience causing activity-induced memory formation  17]. Th. Th17]. These three studies used different strategies to identify dendritic mRNAs in term of abundance, relative enrichment, and ribosome binding. In particular, the strategy used by Ainsley et al. differed significantly from the other two studies in that they used transgenic mice overexpressing Rpl10a and analyzed selective mRNAs that bound to ribosomes. Due to this difference, it may be supposed that the overlap of dendritic mRNAs between Ainsley et al. and the other two studies may be smaller than that between the other two. However, this was not the case a, suggesN-methyl-d-aspartate (NMDA) receptor activity, ion channel activity, spine growth and maintenance, synaptic plasticity, LTP, and learning and memory. In addition, their dysfunctions were associated with a variety of neuropsychiatric disorders such as fragile X syndrome, Alzheimer’s disease, Down syndrome, schizophrenia, Parkinson’s disease, and autism spectrum disorder (ASD)  . In ordeer (ASD) b,c.ASH mRNA, localized at the tip of daughter cells, requires ribosomes composed of specific ribosomal protein paralogs [Escherichia coli in several studies [The fact that many mRNAs encoding ribosomal proteins were found in the dendritic mRNAs was surprising. It is well known that ribosome subunits are assembled in the nucleus, where ribosome assembly requires many steps and with the help of various regulatory factors . The resparalogs . Given t studies ,204,205, studies . Thus, tEef1a1, Eef1b2, and Eef2 mRNAs. Many translational regulators are known to be recruited to RNA granules as proteins and transported to dendrites [The GO category “elongation factor” included endrites ,196. In endrites . In addiendrites ,69. IncrThe other GO categories “synapse”, “dendritic spine”, and “ionotropic glutamate receptor complex” included mRNAs encoding postsynaptic proteins such as Arc, Kcnab2, Crtc1, Ncs1, Camk2a, Cdk16, Shank1, Homer2, Dlg2, Dlg4, Dlgap3, Sipa1l1, and Psd3 b,c. MostOf note, Homer2 and Psd3 were also classified as proteins that contain membrane-bound pleckstrin-homology (PH) domain in the GO enrichment analysis, although the Benjamini value (0.0535) did not reach significance. The GO categories of “PH domain” and/or “PH-like domain” included Homer2, Psd3, Psd, Plekhm2, Sptbn2, and Arhgap25. Compared with soluble proteins that diffuse easily in the cytosol, membrane-associated proteins are difficult to diffuse freely due to physical obstacles and electrostatic interactions within the membrane . PH domaRNA-binding proteins in RNA granules play a variety of roles in regulating mRNA stability, localization, and translation in neurons. Among them, we focus on five RNA-binding proteins: fragile X mental retardation protein (FMRP), RNG105/Caprin1, Ras-GAP SH3 domain binding protein (G3BP), cytoplasmic polyadenylation element binding protein 1 (CPEB1), and staufen double-stranded RNA binding proteins 1 and 2 (Stau1 and Stau2), of which associated mRNAs have been relatively extensively identified, and as summarized below, their knockout (KO)/knockdown (KD) in rodents are known to affect neural functions and higher-order brain functions, including memory formation.Fmr1 gene, in which mutations cause fragile X syndrome, a mental retardation disorder. FMRP regulates protein-synthesis-dependent synaptic plasticity, because Fmr1 KO mice exhibited enhanced metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) that requires protein synthesis [Fmr1 KO mice was both impaired [FMRP is encoded by the ynthesis ,211,212.impaired ,214,215 impaired . Spatialimpaired ,218 and impaired ,219,220.RNG105/Caprin1 is responsible for the formation and/or maintenance of dendrites and synaptic connections  and is eG3BP plays a central role in the assembly of stress granules (SGs) in response to various types of stress ,224. G3BCPEB1 binds to cytoplasmic polyadenylation elements (CPEs) within the 3’UTR of mRNAs and mediates cytoplasmic polyadenylation of mRNAs to promote translation . CPEB1 rDrosophila embryo development, whereas its orthologs are known to regulate synaptic plasticity in rodents: Stau1 and Stau2 are required for L-LTP and LTD, respectively [Staufen is well known for its role in mRNA localization during ectively ,230. Altectively , Stau2 dectively . In addiectively . Downregectively .To better understand the mechanistic basis of the RNA-binding-protein-mediated regulation of biological functions, including brain functions, it is important to identify target mRNAs for RNA-binding proteins. Various methods have been developed for this purpose ,235.In one method, RNA–protein direct binding in vitro has been analyzed using systematic evolution of ligands by exponential enrichment (SELEX). In SELEX, binding cycles between RNA-binding proteins and RNA pools are repeated to select high-affinity RNAs, and these RNAs are amplified by reverse transcription PCR, which detects RNAs that bind to proteins with high probability ,237,238.Second, RNA–protein association in cells and tissues has been identified with RNA-immunoprecipitation (IP), UV-crosslinking IP (CLIP), and electrophoretic mobility shift assay (EMSA). In CLIP, UV irradiation of cells or tissues generates covalent bonds between RNA and RNA-binding proteins when they are in close proximity to each other, followed by co-IP of the RNA with the RNA-binding proteins ,240,241.Third, RNA-protein association has also been identified using gene overexpression, KD, and KO of RNA-binding proteins in cells and animals. These altered expression levels affect the stability, localization, and translation efficiency of the target mRNAs depending on the effect of the RNA-binding proteins on the mRNAs. Differential analysis of these changes compared with control cells and animals, using microarrays, RNA-seq, ribosome footprints, and their combination with the isolation of specific regions of cells, can identify mRNAs regulated by RNA-binding proteins. There are also various other methods to analyze RNA-protein associations, such as sequence- and structure-based methods using computational approaches .Using these methods, target mRNAs of RNA-binding proteins in RNA granules have been identified. We comprehensively list the target mRNAs of FMRP, RNG105/Caprin1, G3BP, CPEB1, Stau1, and Stau2 and summarize the effects of the RNA-binding proteins on the mRNAs . FurtherFMRP target mRNAs have been identified in many studies and are listed in In contrast, RNG105/Caprin1, for which target mRNAs are listed in The association of G3BP with mRNA has been analyzed in several studies, including comprehensive identifications of G3BP-associated mRNAs  362,363,371,372.Each RNA-binding protein has its own functions that regulate the expression, stability, localization, and translation of its target mRNAs. In addition to executing these intrinsic functions, RNA-binding proteins in RNA granules may also play roles in regulating mRNAs through controlling the condensation and dissolution of RNA granules. Recently, it has been revealed that RNA granules are formed by liquid–liquid phase separation and their liquid-like and solid-like states can be dynamically controlled ,380,381.Camk2a mRNA localization to dendrites has been reported to be required for L-LTP and long-term memory formation. This was revealed by the deletion of the 3’UTR, which caused a loss of dendritic localization of Camk2a mRNA but did not affect its translation [The functions of RNA-binding proteins have been revealed in many studies. In addition, their target and dendritic mRNAs have been identified, and the functions of the proteins encoded by these mRNAs have been elucidated. However, there is limited understanding of the physiological relevance of locally increasing the concentration of mRNA-encoded proteins through mRNA localization and local translation. Exceptionally, nslation . However


Malignant melanomas (MMs) were the fifth most common cancer in men and the sixth most common cancer in women in 2018, respectively. These are characterized by high metastatic rates and poor prognoses. We systematically reviewed safety and efficacy of carbon‐ion radiotherapy (CIRT) for treating MMs. Eleven studies were eligible for review, and the data showed that MM patients showed better local control with low recurrence and mild toxicities after CIRT. Survival rates were slightly higher in patients with cutaneous or uveal MMs than in those with mucosal MMs. CIRT in combination with chemotherapy produced higher progression‐free survival rates than CIRT only. In younger patients, higher rates of distant metastases of gynecological MMs were observed. The data indicated that CIRT is effective and safe for treating MMs; however, a combination with systemic therapy is recommended to ensure the best possible prognosis for MMs. The purpose of this study is to systematically review the safety and efficacy of carbon‐ion radiotherapy (CIRT) for malignant melanoma. Eleven studies were eligible. For overall survival, cutaneous melanoma, and uveal melanoma had a slight advantage than mucosal melanoma. Concurrent CIRT with chemotherapy exhibited superior PFS than CIRT alone. Younger patients presented a higher distant metastasis for gynecological melanoma. Although available data demonstrated that malignant melanoma is effectively and safely treated with CIRT, it is suitable to organically combine systemic therapy to obtain the best prognosis for melanoma patients. Depending on their location, MMs can be classified as cutaneous, uveal or mucosal. Previous studies suggest that average morbidity rates are 16.2 per 100 000 in the United States and 0.383 per 100 000 in Japan.Carbon‐ion beams produce increased energy deposition at the end of their range to form the Bragg peak while minimizing irradiation damage to the surrounding tissues, which facilitates precise dosage and localization, compared with photon beams. Furthermore, carbon‐ion beams are cell cycle independent with a high relative biologic effectiveness (RBE) and low oxygen enhancement ratios.22.1This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) statement, and the review protocol is available in PROSPERO (CRD42019141495). All literature searches were conducted until October 15, 2019, using the search tools of Embase, Cochrane Library, Web of Science, and PubMed with the search terms “hadron”, “particle”, “charged particle”, “heavy ion*”, OR “carbon ion”, AND “melanoma”. In order to include as many eligible studies as possible, one last duplicate search was performed on March 5, 2020. Only publications written in English were considered. Additional studies were identified from citations in conference abstracts, review articles, and reference lists. All references were screened to ensure that relevant studies were included.2.2Studies were included if they matched the following criteria: (a) clinical or retrospective studies reporting effectiveness and/or adverse effects on patients with MMs who were treated with carbon‐ion beams; (b) Trials enrolling adults; and (c) tumors had been diagnosed by histopathology. Publications were excluded if they were (a) studies only reporting photons, protons or other heavy particles; (b) case reports on one or two patients; (c) letters, editorials, protocols, reviews, or meta‐analyses; (d) duplicate publications; (e) cell and animal experimental studies; (f) lacking detailed data.2.3The necessary data was extracted by two investigators independently, and results were discussed with senior investigators until a consensus was reached. The primary outcome was OS, and secondary outcomes were local control (LC), progression‐free survival (PFS), treatment‐related toxicity, local recurrence, and distant metastases (DM). The following data of each article were processed: first author, publication year, study design, number of patients, institution, study period, tumor site, tumor status, total treatment dose, and median follow‐up time.2.4Case series reports were evaluated using the case series report bias evaluation tool in Table 33.1As shown in Figure 3.2Data regarding tumor status, treatment planning, OS, LC, other therapies, local recurrence, and DM are shown in Table P = .041).In addition to the reports mentioned above, two retrospective studies reported diverse outcomes of CIRT used for treating gynecological MMs. The 5‐y OS and LC rates were 28% and 44%. DMs occurred in 45% and were more frequent in the younger age group (<71 years) than in older patients , 1‐, 3‐, and 5‐y OS rates of MMs are 63%, 30%, and 20% in Europe.Compared with photons and protons, carbon‐ion beams directly cleave double‐stranded DNA at low oxygen concentrations and emit lower radiation doses to the surrounding healthy tissue, resulting in better therapeutic ratios. CIRT was first applied in MMs in 1994. Results showed 3‐y LC rates above 80%,Among the important clinicopathological factors of MMs, five independent predictors of mortality have been identified, and depth of primary tumors, termed T stage, is the most important factor. Metastasis is an important predictor of mortality.50 of the iris‐ciliary body were also found to be associated with lower incidences of neovascular glaucoma.Uveal MMs are characterized by high local control (90%), high metastasis rates (50%), and low survival. Nucleation, radiation with brachytherapy, and proton RT were the most common treatment methods.MMs are mostly of cutaneous origin; however, 18% of MMs occur in the female genital tract. The most common site of gynecological MMs is the vulva (70%), followed by the vagina and the lowest incidence in the cervix. In general, the prognosis of gynecological MM is poorer than that of non‐gynecologic MMs. Briefly, 5‐y OS were 18.8%, 11.1%, and 0% for stage I, stage II, and stages III‐IV, respectively.During the early 21st century, CIRT treatments for various malignancies have been initiated in China, and patients with MM achieved promising survival rates.It has been reported in several studies that 3.2%‐29% and 3%‐14% of patients with H&N MMs developed acute and late skin or mucosal toxicity of G3 or higher after photon, fast neutron, or proton RT, respectively. Especially, tumors close to critical anatomic structures show higher incidence of toxicity.BRAF (40%‐70%) and NRAS (15%‐30%); C‐KIT mutations showed the lowest prevalence (5%‐10%).Tumor recurrence and metastasis are closely associated with the biological characteristics of tumor cells. Although surgery, RT, chemotherapy, immunotherapy, and targeted biological therapies are frequently used to treat MMs, they do not produce particularly strong effects with respect to controlling recurrence and metastasis. Local recurrence and DM rates were 29%‐60.7% and 29%‐76%, respectively, in patients treated with surgery only or with surgery plus postoperative RTOur review had several limitations. Firstly, no relevant randomized clinical trial data are available due to present scarcity of carbon‐ion RT equipment and low incidence of MMs, while surgery remains the mainstay of treatment options. All studies reviewed here were clinical series reports with small sample sizes. Secondly, there is a bias in patient characteristics . In addition, OS, LC, toxicity, recurrence, and DM data were not consistently available because of incomplete follow‐up reporting. Furthermore, most patients included in the reviewed studies suffered from unresectable or relapsed tumors, whereas randomized controlled trials of other treatment modalities for comparable cases are lacking. These limitations therefore may affect reliability of the drawn conclusions. However, the following observations seem highly likely: (a) CIRT produces better LC with low recurrence and toxicity effects in MM treatments, compared to surgery, conventional photon RT, or proton RT; (b) despite better LC, CIRT still shows unsatisfactory OS rates. However, we believe that this therapy produces relatively good results, compared with those of other treatment options. This hypothesis is supported by the fact that almost all CIRT‐treated patients included in the reviewed studies had suffered from inoperable or recurrent tumors, and CIRT substantially reduced local recurrence. (c) In terms of OS after CIRT treatments, cutaneous and choroidal MMs showed better results than mucosal MMs. Among mucosal MMs, OS was lowest in patients with gynecological MMs. (d) CIRT concurrent with chemotherapy exhibited superior PFS compared to CIRT alone. (e) Patients younger than 71 years showed a higher incidence of DM during CIRT treatments of gynecological MMs. (f) Toxicity apparently did not differ between CIRT with and without DAV therapy, aside from hematologic adverse events. (g) Compared with surgery and conventional photon RT, CIRT seems to be a more promising option for patients with inoperable and recurrent tumors and tumors adjacent to important organs.5CIRT is a safe, effective, and feasible treatment option for MMs, particularly for patients with tumors close to critical anatomical structures or with recurrent tumors. Future efforts to improve prognoses should focus on both metastasis and survival. More effective systemic therapy options to be combined with CIRT are necessary.The authors have no conflict of interest.Chengcheng Li finished the manuscript; Xiaohu Wang and Qiuning Zhang contributed to choose research directions. Zheng Li and Kehu Yang assessed the level of evidence; Ruifeng Liu and Hongtao Luo searched electronic database; Yichao Geng, Lina Wang, Shuangwu Feng, Xueshan Zhao and Zhen Yang contributed to screened literatures. Qiang Li revised the final manuscript.The data used to support the findings of this study are available from the corresponding author upon request. No ethic approval was required as all data originated from previously published studies.


Our results suggest that oligomerization plays a key role in regulating RNA DEAD-box helicase activity.Members of the DEAD-box helicase family are involved in all fundamental processes of RNA metabolism, and as such, their malfunction is associated with various diseases. Currently, whether and how oligomerization impacts their biochemical and biological functions is not well understood. In this work, we show that DDX21, a human DEAD-box helicase with RNA G-quadruplex resolving activity, is dimeric and that its oligomerization state influences its helicase activity. Solution small-angle X-ray scattering (SAXS) analysis uncovers a flexible multi-domain protein with a central dimerization domain. While the Arg/Gly rich C termini, rather than dimerization, are key to maintaining high affinity for RNA substrates,   •The human RNA DEAD-box helicase DDX21 is dimeric•DDX21 dimerization is mediated by a hydrophobic central core domain•SAXS-based modeling reveals that DDX21 is intrinsically flexible•Dimerization and C-terminal domains mediate G-quadruplex and dsRNA unwinding Molecular Modelling; Biological Sciences; Molecular Biology; Structural Biology RNA DEAD-box helicases perform structural rearrangements of RNA and RNA/protein complexes in many cellular processes including ribosome biogenesis, transcription, translation, and RNA editing . Their nbona fide helicase able to unwind dsRNA, can also resolve other nucleic acid structures like R-loops or RNA G-quadruplexes . Comparison of the electrostatic potential distribution on the surface of the GUCT/RRM domains from different enzymes might suggest they have different functions and will be discussed later in the context of RNA binding.Previous work has shown that in addition to the DEAD-box HC, DDX21 contains a Gu C-terminal (GUCT) domain and a C-terminal basic tail  and several N- and C-terminal truncation constructs were expressed and purified  (residues 568–620) B. The tepurified C, they bpurified D. On thepurified D. DDX21Feriments .Fl and DDX21ΔN mutants were generated in which the conserved hydrophobic residues in the α4 helices (residues 611–616 [LAAALA]) were mutated to charged arginines, to create repulsion upon dimerization  is mainly monomeric , the second peak to the protein dimer (216 ± 21 kDa), and the third peak to a protein dimer bound to two R-loop molecules (302 ± 33 kDa)  C and S6.These experiments suggest that DDX21 is a dimer in the apo conformation as well as in complex with RNA substrates. DDX21 appears therefore structurally homologous to the bacterial enzymes  and dimerizes via a small hydrophobic helical domain. Inspection of its DD amino acid composition suggests that the DDX21 paralog DDX50 might also be a dimer B and S7.Fl binds RNA in the absence of ATP with the highest affinity for the RNA G-quadruplex (Q2 RNA)  and a ∼3-fold decrease to the RNA G-quadruplex binding. These results indicate that this domain, most likely unstructured (see below), makes only a small contribution to RNA binding. On the other hand, deletion of the C-terminal basic tail significantly reduces the affinity by at least one order of magnitude for all substrates (DDX21ΔNC). The additional deletion of the GUCT domain in the DDX21Core-DD construct slightly recovers this drop (by 1.5-fold), still remaining far below the DDX21Fl binding affinity values. DDX21Core is the weakest binder, whereas the monomeric DDX21 mutants bind RNA as tightly as the wild-type protein, emphasizing that the C-terminal basic tail is the key for RNA recognition, whereas dimerization seems not to be essential.Using the acquired structural information, we sought to understand the influence of dimerization and each accessory domain in RNA binding and unwinding. The binding affinities of the DDX21 variants to ssRNA, dsRNA, and RNA G-quadruplex were measured using fluorescence polarization experiments  2.6 nM , whereas) 2.6 nM A and 3B.in vitro helicase assay  data and flexible fitting of individual domain structures is a powerful tool to understand the conformation and the structure-function relationship in these multi-domain systems . We, theg) of 70 Å for Fl, 48 Å for ΔN, 46 Å for ΔNC, and 44 Å for Core-DD. The large difference between the Rg value of the DDX21Fl and the rest of the constructs suggests the presence of a core globular structure and an elongated flexible N terminus. This is apparent when comparing the pair distance distribution functions P(r) and the maximum dimensions (Dmax) for the DDX21Fl and the variants in Fl shows a profile characteristic of elongated structures with a very large Dmax of 319 Å and a main peak at a much shorter radius (dark blue in max of about 177 Å (orange in Fl (dark blue). As a reference, compact, globular molecules show a peak at the Guinier-Kratky point  where the dashed lines in SAXS was performed coupled to size exclusion chromatography (SEC-SAXS) to avoid aggregation effects . The GuiCore  function in Core-DD does not form the “inverted V-shaped” structure as seen, for example, in the CshA crystal structure  = 62°C versus Tm (ΔNC) = 38°C, Compared with the compact globular shape previously described for the monomeric DDX21Core , DDX21Cotructure   and 5' single-stranded overhangs  in an ATFl binds RNA G-quadruplex with the highest affinity, when compared with other RNA structures, and this preference already exists in the DDX21Core-DD and DDX21Core constructs. Interestingly, the bacterial DEAD-box helicase CsdA also shows the same behavior, given that the DD-Core dimer of CsdA has specificity for the G-rich substrates over other RNA structures tested . This indicates that oligomerization cannot be overlooked when studying the mechanism of action of this helicase family. Further evidence on the role of oligomerization comes from the association of other helicases in cells .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Matteo Dal Peraro (All unique/stable reagents generated in this study are available from the Lead Contact without restriction.SAXS data and models were deposited in the Small Angle Scattering Biological Data Bank SASBDB with accession codes SASDGU9, SASDGV9, SASDGW9, SASDGX9, and SASDGY9.All methods can be found in the accompanying


The human immunodeficiency virus type 1 (HIV-1) proteome is expressed from alternatively spliced and unspliced genomic RNAs. However, HIV-1 RNAs that are not fully spliced are perceived by the host machinery as defective and are retained in the nucleus. During late infection, HIV-1 bypasses this regulatory mechanism by expression of the Rev protein from a fully spliced mRNA. Once imported into the nucleus, Rev mediates the export of unprocessed HIV-1 RNAs to the cytoplasm, leading to the production of the viral progeny. While regarded as a canonical RNA export factor, Rev has also been linked to HIV-1 RNA translation, stabilization, splicing and packaging. However, Rev's functions beyond RNA export have remained poorly understood. Here, we revisit this paradigmatic protein, reviewing recent data investigating its structure and function. We conclude by asking: what remains unknown about this enigmatic viral protein? HIV infects approximately 37 million people globally and is treated using life-long anti-retroviral therapy (ART) . I. Itat gerize (b) . A–A andxibility . The disxibility ,14,15. N2.2.a) [figure 1a) [rev and the other fully spliced mRNAs are exported to the cytoplasm as they are recognized as ‘mature’ transcripts [Kd = 0.3–5 nM), using the arginine residues of the ARM motif aligned in an α-helix which are projected to and inserted into a major groove in the RNA (b) [c). These sites are separated by approximately 55 Å, which matches the span of a Rev dimer [Following reverse transcription, HIV-1 proviral DNA integrates into the host cell genome. Production of HIV-1 RNAs is then mediated by the host RNA polymerase II giving rise to over 100 different RNA species by alternative splicing . The appfigure 1a) . The appgure 1a) . In the gure 1a) . Conversnscripts ,20. Oncenscripts ,22. Ribonscripts ,24. In tnscripts –33. Rev  the RNA b [29,31, RNA (b) –37. How  RNA (b) ,36,38–42 RNA (b) ,34,43. T RNA (b) , where t RNA (b) ,45, mapsev dimer . It has ev dimer . Similarev dimer . A diffeev dimer . This moev dimer . Nucleotev dimer . Accordiev dimer ,43,48,49ev dimer ,50.d), which has been shown to occur exclusively following RNA dissociation [After Rev binds the RRE, host cell exportin Chromosome Region Maintenance Gene 1  and RanGTP are recruited to the Rev NES ,52. Rev figure 2ociation . In this2.3.et al. [rev use TAP/NXF1 to be exported, accumulation of Rev protein may lead to the suppression of TAP/NXF1 export pathway to increase the ratio of underspliced to spliced transcripts in the cytoplasm. Accordingly, Rev was proposed to interfere with the association of TAP/NXF1 components, such as ALY/REF, with RNA [et al. [cap-binding complex (CBC) to inhibit its interaction with Aly/REF and subsequently suppress downstream TAP/NXF1 export. Rev can indeed bind subunits of the nuclear CBC [Rev reduces the levels of cellular and viral RNAs exported by CRM1-independent pathways by blocking TAP/NXF1, the main cellular pathway that exports the bulk of cellular mRNAs. Taniguchi et al.  showed tet al. . This suwith RNA . Althoug [et al.  propose lear CBC , which l2.4.env, gag and pol coding regions and promote nuclear retention, instability and reduced polysome loading of viral RNA [gagpol increases protein expression by ameliorating steady-state mRNA levels [gag and pol genes results predominantly from an increase in cytoplasmic mRNA [In addition to stimulating nuclear export, Rev may serve several post-transcriptional roles to promote HIV-1 expression. For example, Rev binding to the RRE appears to overcome the inhibitory effects of instability (INS) sequences in HIV-1 RNAs. These INS regions have been identified in the iral RNA –69, indeiral RNA –72. The A levels ,74, illuA levels ,76. IndeA levels –68, sincA levels ,74,77. Hmic mRNA –82. Rev 2.5.in vitro and its overexpression can inhibit Rev function and HIV-1 gene expression in a dose-dependent manner [Rev may inhibit splicing in an RRE-dependent manner. The ARM peptide of Rev was found to inhibit splicing of RRE-containing RNA up to 15-fold more than control RNA lacking the RRE . Incubatt manner . It alsot manner –89. It it manner . Hence, t manner , alignin2.6.gag mRNA and protein levels in the absence of Rev [env RNA associated with monosomes when infected with HIV-1 lacking Rev (HIV-1Rev(-)), whereas over 75% associated with polysomes when infected with wild-type Rev HIV-1 [tat was unaffected by the presence or absence or Rev, indicating efficient assembly of ribosomes onto these viral RNAs in a Rev-independent manner. Interestingly, Rev-dependent association of underspliced viral RNA with polysomes was found to be dependent on the presence of the RRE, suggesting the need of Rev/RRE interaction for this phenomenon to occur [in vitro translation [It has been proposed that Rev influences the translation efficiency of viral RNA. Early research highlighted disproportionate increases in viral Envelope protein expression relative to total mRNA levels in Rev and Tat-transfected cell lines . Additioe of Rev –96, provev HIV-1 . Conversto occur . Howeverto occur . These rto occur . Rev enhnslation . Howevernslation . Whether2.7.Rev(-) expression plasmid [Rev(-) genomic RNA including MS2 stem-loops was co-transfected with MS2-TAP [The presence of a packaging signal in the 5′ untranslated region (UTR) of HIV-1 genomic RNA does not suffice for efficient assembly of the viral RNA into viral particles. The interaction of Rev with the RRE is also proposed to contribute to viral RNA packaging –104. The plasmid . They re plasmid . However MS2-TAP . This su MS2-TAP . The mec MS2-TAP .The Rev-binding site situated in the HIV-1 5′ UTR (loop A of SL1) is also proposed to stimulate viral RNA assembly into viral particles ,100,108.2.8.2+ was recently reported to impact the structural dynamics of the RRE and potentially Rev binding [HIV-1 cannot encode all the machinery required for its replication and spread. Thus, it is heavily reliant on host cell resources. One prevalent strategy used by viruses to hijack cellular resources is to express viral proteins that interact with and recruit key cellular factors. By knowing which cellular factors Rev interacts with, it is thus possible to obtain deeper mechanistic insights into its regulatory roles. A common approach for discovering protein–protein interactions (PPIs) globally involves immunoprecipitation (IP) followed by mass spectroscopy analysis (MS). This approach has been employed to reveal the interactomes of HIV-1 proteins –114. Few binding . It has  binding , which m binding ,121,122.Despite the challenges of uniting various interactome data, there is a set of proteins identified consistently in a number of different studies, imbuing them with credibility ,123,124.gagpol mRNA in the cytoplasm transcribed from a Gagpol-RRE expression vector, confirming that it influences Rev-dependent RNA export [DEAD (Asp–Glu–Ala–Asp)/H box RNA helicases have been functionally implicated in all steps of RNA metabolism as well as ATP-dependent RNA duplex unwinding. Several DEAD/H box proteins interact with Rev, most prominently, DDX1. DDX1 was initially identified to bind a motif of Rev in yeast-two hybrid screens and then confirmed to interact with Rev using protein overexpression and co-immunoprecipitation ,125. DDXA export . Other hA export ,128. It et al. [et al. [CRM1 was one of the earliest Rev interactors identified. It was initially shown to bind Rev through separation of nuclear envelope proteins by gel electrophoresis, followed by treatment with radiolabelled Rev protein , which ret al.  expressiet al. . Interes [et al.  found thEukaryotic Initiation Factor 5A (eIF5A) is an essential protein that facilitates translation elongation of polyproline regions and prevents ribosomal stalling . It can 2.9.Rev is critical for HIV-1 gene expression and, therefore, represents a potential anti-viral target. Despite this, there are currently no Rev-based therapeutics in clinical use. It has long been known that dominant negative mutants of Rev can abrogate wild-type Rev function in lymphocytic cells ,141. ForOther therapeutic approaches target the Rev/RRE interaction itself using small molecules. An early iteration of this approach used aminoglycoside antibiotics which specifically bound to the RRE at the Rev-binding site , blockinet al. [As CRM1 is required to escort RNA-bound Rev across the nuclear envelope, blocking the CRM1/Rev interaction is a potential anti-viral opportunity. It is well established that CRM1-inhibiting drugs, such an anti-fungal agent leptomycin B, can restrict HIV-1 replication ,164. Howet al. ). Therefet al.  and elicet al. .et al. developed ABX464 [in vivo. This highlights the importance of reproducibly defining the Rev interactome: it may unlock dozens of potential therapeutic targets.Targeting other Rev–host cofactor interactions is similarly difficult but not impossible: Campos d ABX464 , a drug d ABX464 . Importad ABX464 . It has 3.3.1.et al. [et al. [The role of Rev in RNA trafficking was identified shortly after its discovery in 1986; in the subsequent 30 or more years of research, Rev has continued to pose more questions than answers. For example, we have discussed here the known cofactors of this elusive protein, though the reader may note that much of this research is ‘correlative’ with the underlying molecular mechanism remaining unknown. As Rev is a difficult protein to study , the numet al.  are also [et al. . However3.2.et al. [While Rev participates in nucleocytoplasmic shuttling, it also displays a well-documented tendency to localize in the nucleolus ,168,169,et al.  have shoet al. ,168, suget al. . Additioet al. . Howeveret al. . The rol3.3.Some of the challenges of working with Rev have been solved thanks to recent technological advances. For example, Rev tends to aggregate and precipitate , and the4.unknowns. These improved approaches will hopefully advance us towards understanding the structure, functions and interactions of the HIV-1 Rev protein. In turn, a better understanding of Rev biology will probably open novel therapeutic avenues on the quest to combat HIV-1 infection.Much is known about the HIV-1 Rev protein, particularly of its essential role in RNA export. However, many questions remain unsolved. It is thus vital that systems and methodologies are established which strive to more closely emulate natural HIV-1 infection and native Rev activity when elucidating the


Metastasis is the prominent cause of death in patients with hepatocellular carcinoma (HCC); however, the mechanisms behind HCC metastasis are not well understood. MicroRNAs (miRs) can regulate gene expression and affect HCC metastasis. Exosomes can transport miRs and other cargoes to and from different cells, thus being associated with tumour-distant metastasis. Exosomal miRs involve different processes of HCC metastasis through their functional effects, such as their induction of epithelial-to-mesenchymal transition, angiogenesis, and distant niche. In this review, data from the literature were analysed and summarised, with a focus on the evidence extraction of exosomal miRs in HCC metastasis with the purpose of increasing the understanding of the mechanisms behind HCC metastasis and acquiring implications for application. Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-associated death and the sixth incidence of malignancy worldwide, accounting for ~780,000 deaths and 840,000 new cases in 2018 . MalignaExosomes are small extracellular vesicles (EVs) that are released from cells on the fusion of an intermediate endocytic compartment, the multivesicular body, with the plasma membrane . ExosomemiRs are evolutionarily conserved, endogenous, noncoding single-stranded RNA molecules of 18–25 nucleotides. miRs effectively regulate gene expression by inhibiting the translation of target mRNAs . It is eExosomes are small EVs  released by various cell types and rich in functional molecules such as lipids, proteins, mRNA, and miRs , 29. ExoFocal adhesion kinase (FAK) has been reported as being associated with cancerogenesis and the development of HCC, and miRs may modulate cancer cell migration and metastasis by influencing FAK-facilitated cytoskeletal rearrangement, as well as stroma adhesion reconstruction . For exaTian et al.  reportedA hypoxic tumoural microenvironment can induce angiogenesis and proliferation of tumour , 41. In Blood-borne metastasis is the primary cause of mortality in patients with cancer and increased vascular permeability facilitates metastasis . A studyHistone deacetylase 6 (HDAC6) has a crucial role in multiple cellular processes, and the dysregulation of HDAC6 may be associated with the progression of some cancers . In the β1-integrin-NF-κB signalling pathway by downregulating β-1,4-galactosyltransferase III, thus transforming fibroblasts into CAFs. CAFs promoted metastasis by secreting IL-6 and IL-8 and proinflammatory cytokines that enhanced stemness and EMT in HCC cells. In addition, the serum exosomal miR-1247-3p levels in HCC patients with lung metastases were notably more elevated than those in patients without lung metastases. These data suggest that HCC cell-derived exosomal miR-1247-3p can promote an inflammatory microenvironment and lung metastasis.As the most prolific cell type in the cancer microenvironment, CAFs are a subset of transformational fibroblasts that facilitate tumour progression and metastasis . Fang etIn a study by Zhang et al. , CAFs anHSCs are the major interstitial cell type in the HCC microenvironment and an important component of invasion and metastasis . A studyCSCs may be the result of the malignant transformation of normal stem cells that have stronger self-renewal ability and metastatic potential , 51, andStudies have shown that MCs are abundant in the cancer microenvironment . For exaExosomes are critical mediators of intercellular communication, through which miRs are transported between various different cells. The high expression of oncomiRs and low expression of tumour suppressing miRs can result in the formation of metastatic foci. The occurrence of metastasis depends not only on exosomal miRs secreted by HCC cells but also on a variety of other cells in the HCC microenvironment. In the HCC microenvironment, by transferring exosomal miRs, HCC cells can activate the conversion of fibroblasts, HSCs, and even normal liver cells into cancer-related cells, resulting in malignant phenotypes. This can also weaken the integrity of endothelial cell connectivity and increase vascular permeability. In turn, exosomal miRs derived from activated HSCs, fibroblasts, and CSCs can promote HCC metastasis.HCC metastasis is accompanied by the abnormal expression of exosomal miRs, and the detection of this aberrant expression can be used to evaluate HCC metastasis and prognosis. Exosomal miRs that promote or inhibit HCC metastasis may be used for prevention and treatment. Tian et al.  developeAfter more than two decades of research, great progress has been made in the field of exosomal biology; however, it is still considered to be in its infancy, and the role of exosomes in tumour progression requires further investigation. Advancement in understanding of exosomal miRs will make them become promising candidates for the diagnosis and treatment of HCC.