Most studies of ncRNAs in plants have focused on the identification of silencing microRNAs (miRNAs) and small interfering RNAs (siRNAs). Another important family of ncRNAs that has been well characterized in plants is the small nucleolar RNAs (snoRNAs) and the related small Cajal body-specific RNAs (scaRNAs). Both target chemical modifications of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In plants, the snoRNA genes are organized in clusters, transcribed by RNA Pol II from a common promoter and subsequently processed into mature molecules. The promoter regions of snoRNA polycistronic genes in plants are highly enriched in two conserved In order to identify novel ncRNA genes, we have used the snoRNA Telo-box/Site II motifs combination as a functional promoter indicator to screen the Arabidopsis genome. The predictions generated by this process were tested by detailed exploration of available RNA-Seq and expression data sets and experimental validation. As a result, we have identified several snoRNAs, scaRNAs and 'orphan' snoRNAs. We also show evidence for 16 novel ncRNAs that lack similarity to any reported RNA family. Finally, we have identified two dicistronic genes encoding precursors that are processed to mature snoRNA and miRNA molecules. We discuss the evolutionary consequences of this result in the context of a tight link between snoRNAs and miRNAs in eukaryotes.We present an alternative computational approach for non-coding RNA detection. Instead of depending on sequence or structure similarity in the whole genome screenings, we have explored the properties of promoter regions of well-characterized ncRNAs. Interestingly, besides expected ncRNAs predictions we were also able to recover single precursor arrangement for snoRNA-miRNA. Accompanied by analyses performed on rice sequences, we conclude that such arrangement might have interesting functional and evolutionary consequences and discuss this result in the context of a tight link between snoRNAs and miRNAs in eukaryotes.The online version of this article (doi:10.1186/s12864-015-2221-x) contains supplementary material, which is available to authorized users. The synthesis of functional transcripts in eukaryotes always involves non-coding RNAs (ncRNAs) within ribonucleoprotein complexes (RNPs) that direct the processing of larger precursor transcripts. Two major families of such molecules include snRNAs and snoRNAs, which direct the splicing and guide the chemical modification of RNAs, respectively . In addiIn silico approaches represent another important strategy for the identification of novel ncRNAs in a variety of sequenced genomes. Most of these techniques use algorithms that consider structural RNA features that are conserved in known RNA families. Here, we propose a distinct approach that employs conserved promoter elements of plant snoRNA genes for the identification of novel ncRNAs in the Arabidopsis genome.In the past decade, a major goal in plant biology research has been to identify novel ncRNAs that regulate cell growth, development or adaptation to biotic and abiotic stresses. Deep sequencing of size-fractionated RNAs has become a major source of ncRNA discovery, producing myriad ncRNA candidates . The chaThe snoRNAs represent an abundant family of ncRNAs found in the nucleoli of all eukaryotes. Most snoRNAs belong to one of two subclasses, C/D box snoRNAs and H/ACA box snoRNAs, which guide 2’-O-ribose methylation and the pseudouridylation of specific RNA targets, respectively . The C/DFunctionally similar to snoRNAs are scaRNAs, which direct RNA modifications of snRNAs in Cajal bodies. scaRNAs are larger than the predominant classes of snoRNAs and possess the characteristic boxes of both C/D and H/ACA snoRNAs as well as CAB boxes (UGAG), which function as Cajal body localization signals. Similar to snoRNAs, scaRNAs form conserved scaRNPs with fibrillarin and dyskerin .snoRNAs have distinct targets and functions. Most are responsible for the modification of ribosomal RNAs (rRNAs), but others direct changes in other classes of RNAs, including tRNAs and snRNAs . In addiIn addition to the canonical structured snoRNAs, many others have been discovered that, while maintaining the canonical C/D or H/ACA core structure, have additional extensions and fulfill extra functions. One example is the telomerase RNA (TR), a subunit of the RNP telomerase complex, which can guide the synthesis of telomeres in mammals. TR is a 400- to 500-nucleotide RNA characterized by a 3’ H/ACA snoRNA structure .Furthermore, some snoRNAs have dual functions, as they are processed into small ncRNAs with miRNA-like functions in animals –15. In pIn Arabidopsis and rice, more than 200 canonical snoRNAs and scaRNAs have been identified so far . Most plTelo-box (AAACCCTA), which has a sequence related to telomere repeats, and the Site II element (TGGGCY) [Telo-box acts synergistically with the Site II element to coordinate the expression of these genes throughout the cell cycle [Notably, in Arabidopsis, nearly all RP genes and other genes encoding proteins related to ribosome biogenesis and translation, together with polycistronic snoRNAs, share two conserved promoter elements: the (TGGGCY) . These t(TGGGCY) . An anal(TGGGCY) . Gain-ofll cycle . Additioll cycle .The overall data strongly indicate that TeloSII promoters coordinate the expression of snoRNAs along with that of protein-coding genes implicated in ribosome biogenesis and in translational control during the cell cycle. The polycistronic organization of snoRNAs would further contribute to the coordinated expression of these molecules in plants.in silico approaches designed to detect novel ncRNAs are mainly based on algorithms considering the structural features of known RNA families. Here, to reduce the noise-related 'RNA background' and to enhance the detection of new RNA families, we propose an approach based on using the TeloSII promoter motifs of the plant snoRNA genes to search for novel ncRNAs. Application of this strategy to the Arabidopsis genome resulted in the discovery of 26 novel snoRNA-like species, 16 novel ncRNAs without any relation to already described RNAs, and two dicistronic snoRNA-miRNA genes. We further show that this arrangement can also be found in rice, suggesting a tight evolutionary relationship between snoRNAs and microRNAs in plants.High-throughput sequencing approaches dedicated to the identification of novel functional ncRNAs produce an enormous amount of putative predictions; in plants, most predictions correspond to repeat-derived siRNAs originating from spurious transcription or RNA degradation. Likewise, cis-regulatory elements in Arabidopsis, we assembled a customized pipeline  were found (regardless of their order and distance) in a sequence window of 1 kb as suggested earlier by Gaspin et al. [To systematically identify novel non-coding RNA (ncRNA) genes directed by TeloSII ine Fig. . We consn et al. . The nexn et al. –24, we sn et al.  and infon et al.  and the n et al. . Finallyn et al. .Fig. 1CoThe first screening identified 3896 non-redundant TeloSII loci in Arabidopsis intergenic regions, of which 3317 loci were placed upstream of the transcription start sites (TSSs) of annotated protein coding genes  or monocotyledonous (Oryza sativa) plant genomes. However, the absence of conservation in the distant species must be taken with caution. The plant snoRNAs exhibit varying degrees of divergence among species according to their targets. Some are well conserved among distant species like Arabidopsis and rice, while others are restricted to closely related ones. However, snoRNAs with different sequences can still be functional orthologs, targeting the same rRNA residue for modification in the two distant species [Comparative analysis with BLASTN and Phytozome database version 10)  revealed0  reveal species , 25. AltTargets for C/D and H/ACA snoRNAs were predicted using the PLEXY  and RNAsFor the remaining three predicted C/D box and three predicted H/ACA box snoRNAs we could not find the target; therefore, following the widely accepted classification they should be rather considered as 'orphan' snoRNAs . For theIn most cases, expression of the predicted snoRNAs/scaRNAs is supported by RNA-Seq data. However, except for ncR20, which is encoded by cluster 7 Table , none haCluster 1 was previously reported to be expressed as individual snoRNAs, represented by independent lincRNAs, rather than as a polycistronic transcript. To confirm our prediction of the clustered expression of these snoRNAs, we amplified the polycistronic precursor by RT-PCR. Our result clearly shows the amplification of one precursor encoding predicted snoRNAs  contains a classical TeloSII motif associated with a TATA box element Fig. . ScreeniSeveral lines of evidence indicate that miR775 and snoR775 are co-transcribed and produced from the same precursor. The first line of evidence is the presence of a 605 nt cDNA BX818024) that encompasses both snoR775 and pre-miR775 Fig. . The sin that encThe identification of this transcript raised a question regarding how snoR775 and miR775 are produced from the same precursor, considering that each RNA uses a different biogenesis pathway. The processing of pre-miRNA depends on the endonuclease DCL1, whereas the processing of polycistronic pre-snoRNAs is initiated by the RNase III homolog of the yeast Rnt1 enzyme . We testThe presence of four miR775 signals ranging from 21 to 24 nt Fig.  is puzzlA similar genomic organization was observed for the sno-miR779 gene Fig. . The TelNo cDNA sequence representing this locus could be found. Therefore, to confirm that the two snoRNAs and the miR779 precursor are co-transcribed, we mapped the transcription start site by 5’ RACE with a primer complementary to 3’ end of pre-miR779. The result showed a major signal mapping upstream of the snoR128 that encompassed snoR128, snoR129 and the pre-miR779 precursor Fig.  & 4b. FuThe identification of these two sno-miRNA genes in Arabidopsis led us to search for a similar arrangement in the rice genome, where TeloSII elements have also been shown to be conserved upstream of RP genes and snoRNA genes . We screosa-miR1850, which has been reported by several groups, has been found to be associated with the rice protein AGO1, and its target transcript Os04g47410) has been experimentally validated . The miR410 has bTelo-box motif. The existence of a cDNA sequence (AK107197) encompassing the whole region suggests that the snoRNAs and miR6250 are co-transcribed and are probably derived from the processing of a common precursor. The presence of single transcript representing the shared precursor was also confirmed by RT-PCR  , 37 and miRNAs appeared later in evolution and are not present in Archaea, but their progression has also been related to TE expansion . InteresWe have identified 60 ncRNAs  in a growth chamber with a 16 h day (150–200 μmol/m/s), a constant temperature of 22°C and a humidity of 70 %. Seedlings were cultivated in 1/2 MS medium under the same conditions as above.Arabidopsis ecotype Columbia-0 wild type and Genomic DNA was extracted from 2-week-old wild-type seedlings using the DNeasy Plant Mini Kit . Total RNA was isolated from 2-week-old seedlings, 3-week-old rosette leaves, and 5-week-old inflorescence tissue using TRIzol reagent . The RNA concentration was measured using a NanoDrop ND-1000 . DNA contamination was removed by digestion using a TURBO DNA-free kit .Reverse transcription (RT) was performed using 3 μg of DNase-treated RNA, an oligo(dT) 18 primer  and SuperScript III Reverse Transcriptase  according to the manufacturers’ instructions. cDNA samples were diluted 5 times, and 2 μl was used as template. In parallel, two additional reactions were carried out without SuperScript RT: one with DNase-treated RNA as a negative control and the other one with genomic DNA (from 2-week-old seedlings) as a positive control. Touch-down PCR amplifications were performed to detect promising candidate ncRNAs using the following thermal profile: 1 cycle of denaturation at 94 °C/1 min, annealing at 65°C/30 s, and elongation at 72 °C/1 min; 29 cycles of denaturation at 94 °C/30 s, annealing at 63 °C/30 s (∆ -0.5 °C/cycle), and elongation at 72°C/1 min; and an additional 10 ~ 13 cycles, depending on the expression level of the gene of interest, of denaturation at 94 °C/30 s, annealing at 53 °C/30 s, and elongation 72 °C/1 min. PCR products were visualized on 1x TBE/2 % agarose gels with 100 bp Plus and 1 kb Plus DNA Ladders  as size markers. Primer sequences can be found in Additional file To examine the termini of transcripts, 5’ and 3’ two-step RACE experiments were conducted with a SMARTer RACE cDNA Amplification Kit  according to the manufacturer’s protocol. PCR reactions were carried out using the Advantage 2 PCR Enzyme System . PCR products were cloned into the pGEM T-Easy vector  and sequenced . The primers are listed in Additional file A total of 30 μg of RNA was separated by denaturing 8 M urea PAGE (15 %). RNA electrophoresis, blot transfer, and hybridization were performed as previously reported . DNA oliArabidopsis thaliana  and Oryza sativa were retrieved from TAIR  [http://rice.plantbiology.msu.edu/) [http://www.mirbase.org/) [http://chualab.rockefeller.edu/gbrowse2/homepage.html) [Arabidopsis lyrata, Capsella rubella, Brassica rapa, Medicagotruncatulaand Oryza sativa were obtained from Phytozome  [http://www.ncbi.nlm.nih.gov/genbank/), and experimentally identified spliceosomal small nuclear RNA sequences were obtained from ASRG (http://www.plantgdb.org/SRGD/ASRG/) [Annotations for is.org/)  and RGAPsu.edu/) , respectse.org/) . Known sse.org/) –24, 27. ge.html) . Genome me.net/) . RibosomD/ASRG/) . rRNA seTelo-box (AAACCCTA) and six associated permutations , Site II element (TGGGCY) and TATA box . Potential open reading frames (ORFs) of novel transcripts were predicted by ESTScan using Arabidopsis parameters [http://rfam.sanger.ac.uk/) [The PatMatch program  was usedrameters . Putativrameters . RNA folrameters . The Rfa.ac.uk/)  was empl.ac.uk/)  for C/D .ac.uk/)  for H/ACArabidopsis thaliana reference genome using Bowtie2 (version 2.2.3) [RNA sequencing data were obtained from various Arabidopsis tissues and plants grown in different conditions   with foln 2.2.3) .First, 500 nt flanking regions of each annotated ath-miRNA were extracted using a homemade perl script. Subsequently, the SnoReport program was used to scan these regions. If any putative snoRNA was predicted and did not overlap with mature miRNA, it was be considered a snoRNA-miRNA candidate. The analysis of homology with other organisms was carried out by using BLAST. For homologous snoRNA detection, the parameters were set as follows: E-value < 1e-5; similarity > 80 %. For homologous miRNA detection, a maximum of two mismatches were allowed.The data supporting the results of this article are included within the article and its additional files.


Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, ALS , and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5–15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation. Ribonucleic acids (RNA) are involved in different biological processes within the cell. The presence of altered, pathogenic variants of RNA often disrupts the normal course of cellular metabolism, what could be caused by an invalid RNA length or structure due to the mutations. Various approaches were tested to inhibit the expression of pathogenic RNA. Antisense oligonucleotides (ASOs) were the first promising tools for this purpose and, over time, ribozymes, decoys, aptamers and short interfering RNAs were developed –4.In 1978, Zamecnik and Stephenson showed for the first time the inhibition of Rous sarcoma viral RNA translation by specific oligodeoxynucleotides . To dateCurrently, there are two major models for the mechanism of ASO silencing . One is Studies of modified oligonucleotides have shown that a synthetic nucleic acid, the gapmer, can efficiently act as an antisense molecule by activating ribonuclease H, followed by mRNA cleavage . A gapmede novo mutations in humans is approximately 1.7. Moreover, certain types of transitions and transversions occur more frequently than others. In the human genome, the most common are C/T (= G/A) substitutions among transitions and G/C (= C/G) among transversions [In a context of genomes, single nucleotide polymorphisms (SNPs) are often imperceptible changes mainly due to a well-functioning DNA repair mechanisms and the degeneracy of genetic code. SNPs are the most common type of genetic variation in the human genome, occurring on average every 100–300 nucleotides. SNPs can be distinguished by transitions and transversions and the transition-to-transversion ratio for versions .Spontaneous or induced DNA and RNA sequence changes can cause diseases, alter the cellular response to drugs or pathogens, and, finally, they may be useful molecular diagnostic genetic markers. Most genetic diseases are polygenic; however, there are known some monogenic units caused by single substitutions that are inherited in a dominant or recessive manner, and may be autosomal or X-linked. The manner of inheritance usually determines the extent of their occurrence within the population. Neurodegenerative disorders such Alzheimer’s or Parkinson’s diseases have rare Mendelian versions that are inherited in a dominant manner, which themselves are not necessarily direct therapeutic targets due to their low incidence. However, they can provide important clues about the underlying disease pathways that function in the population –17. In tHerein, a new approach to improve the performance of SNP selectivity for ASOs is proposed . Based oSCA3 (spinocerebellar ataxia type 3) gene/mRNA, is pathogenic. Particular variants of the G/C SNP cosegregate with the wild type and mutated allele, respectively. Initial experiments were performed on RNA molecules from the fragment of the SCA 3 gene; however, the proposed conception generally refers to heterozygous mutations. Neurodegeneration is a complex process, which can be related to many genes. The particular SNPs were chosen because they are relatively most widespread among analyzed here neurodegenerative disease-causing point mutations as well as for the type of substitution. All of the mutations are inherited in autosomal dominant manner.The targets selected for these studies are fragments of human mRNA, in which point mutations can determine the development of neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease and ALS. The exception analyzed here is the case of Machado-Joseph disease, where the expansion of CAG repeats, and not the targeted G/C SNP of in vitro experiments concerned short, 13-nucleotide mRNA fragments, containing various types of point mutations placed in the center of the selected RNAs. The following SNPs were chosen: (1) C/G transversion in codon 692 of the APP (amyloid precursor protein) mRNA , associated with the development of Alzheimer’s disease; (2) G/C transversion occurring within codon 291 of the SCA3 gene after the CAG trinucleotide repeats ; (3) G/A transition in codon 717 of the APP mRNA , associated with the development of Alzheimer’s disease; (4) A/G transition in codon 693 of the APP mRNA , also associated with the development of Alzheimer’s disease; (5) G/A transition in codon 46 of the SNCA (α-synuclein) mRNA , associated with the development of Parkinson’s disease; (6) G/A transition in codon 53 of the SNCA (α-synuclein) mRNA , associated with the development of Parkinson’s disease; and (7) C/U transition in codon 4 of the SOD1 (superoxide dismutase 1) mRNA , associated with the development of ALS.The first All of the short RNAs , ASO gapmers , ASO inhibitors (7- and 10-mers), and all PCR primers were synthesized on a BioAutomation MerMade12 DNA/RNA synthesizer using β-cyanoethyl phosphoramidite chemistry  and comm2EDTA, pH 7. Only in the case of G/C SNP , buffer containing 10 mM NaCl was used, because of inability to calculate thermodynamic parameters for 100 mM of sodium chloride. Oligonucleotide single strand concentrations were calculated from the absorbance above 80°C and single strand extinction coefficients were approximated by a nearest-neighbor model [M-1 vs. ln(CT/4) plots is within 15% of that derived from averaging the fits to individual melting curves, as expected if the two-state model is reasonable. The statistical analysis of thermodynamic data was carried out using MeltWin 3.5 software. The methods for determining sampling errors in ΔG°37, ΔH°, and ΔS° from the linear regression of TM-1 vs. ln(CT/4) plots using standard statistical analysis has been previously described [M-1 vs. ln(CT/4), dependence was determined. The ΔG°37 value for the averaged 10-4 M concentration was calculated from this dependence and compared with that derived from averaging the fits to individual melting curves. Differences below 15% indicated that a two-state model is reasonable for the melting of the particular analyzed duplex.Oligonucleotides were melted in buffer containing 100 mM NaCl, 20 mM sodium cacodylate, and 0.5 mM Naor model , 23. Absor model . For mosescribed . Based oin vitro transcription from the DNA template and were constructed in the PCR reaction. Two 74-nucleotide long, single-stranded DNA molecules, which were complementary to 18 nucleotides in length, were chemically synthesized and extended in PCR reactions to give 119 base-pairs of double-stranded DNA. The obtained molecule, containing a 99-nucleotide sequence of interest preceded by a T7 polymerase promoter sequence, was used as a template for in vitro transcription with the MEGAshortscript Kit from Ambion.Long RNAs (99-mers) were obtained by 32P ATP using T4 polynucleotide kinase (long RNA) or fluorescein (6-FAM) during the chemical synthesis of the oligonucleotides. Labeled RNA was mixed with ASO in an H-buffer in proportions that were optimized in separate experiments (1:10 for gapmers and 1:1 for inhibitors). The reaction mixture was denatured for 3 min at 80°C and then renatured at room temperature (RT) for 5 min to form duplexes. Next, 0.4 U of RNase H (E. coli) was added and the mixture was incubated for 20 min at 37°C. The time of RNase H digestion and the enzyme concentration were optimized in relation to the fully complementary RNA/gapmer duplexes. Cleavage products were analyzed on a 16% polyacrylamide gel with 7 M urea and then visualized by autoradiography. The digestion efficiency was evaluated from gel images using the MultiGauge 3.0 program (Fuji). The same assay was used to determine the kinetics of RNase H cleavage. A mixture of the appropriate duplex in H-buffer with ribonuclease H was incubated at 37°C and 10 μl aliquots were collected after 30 sec, 1, 5, 10 and 20 min of reaction. Twenty minutes was usually a sufficient time to complete RNA hydrolysis  during chemical synthesis. Each RNA at constant concentration of 1 μM was mixed with ASO gapmer increasing concentration ranging from 10 nM to 1 μM in an H-buffer, containing 20 mM Tris-HCl, pH 7.8, 40 mM KCl, 8 mM MgClHeLa cells were passaged to a 24-well plate and cultured in a standard RPMI 1640 medium containing 10% FBS (Invitrogen), 1x Antibiotic Animycotic solution (Sigma Aldrich), and 1x MEM vitamin solution (Sigma Aldrich). When the confluence reached around 90% per well, the cotransfection of HeLa cells with pEGFP expression plasmid containing cloned fragments of the genes of interest fused to the GFP protein, and gapmer or gapmer and inhibitor molecules, was carried out with Lipofectamine 2000 (Invitrogen), in a medium without antibiotics. After 24 hours of incubation, the cells were washed with phosphate-buffered saline (PBS) and viewed under a fluorescent microscope. Next, for the quantitative analysis of silencing, the total cellular RNA was extracted using the TRIzol® method [5’-GCTGACCCTGAAGTTCATC and GFPr 5’-GCTCCTGGACGTAGCCTTC, resulting in 164 bp product and normalized by human β-actin levels . The qPCR cycles are as follows: 95°C for 5 minutes; (95°C for 10 seconds and 60°C for 1 minute) for 40 cycles.A day before transfection, ® method . TRIzol The statistical analysis of the qPCR results was performed with Bio-Rad CFX Manager 3.0 and OriginLab 8.0 software. Antisense oligonucleotides were tested in various concentration sets, and the transfection of each set was repeated two to five times . Quantitative PCR for each set was repeated three times . The results from the technical and biological repeats for particular samples were gathered for each concentration set transfected with WT or Mut constructs in order to determine the mean relative expression and its standard deviation (Bio-Rad CFX Manager 3.0). Differences between the obtained Cq values for particular repeated concentration sets higher than 0.5 were exluded from the analysis. Next, using ANOVA analysis (OriginLab 8.0), the normalized relative expression of GFP coupled with the WT and Mut RNA variants of analyzed genes was compared at the significance level of 0.05 for particular analyzed conditions. Statistically significant differences of the mean expression between WT and Mut alleles (P<0.05) were marked with an asterisk on the charts presenting qPCR results. Moreover, standard curves for target and reference genes were determined in order to control the PCR efficiency of each reaction, which ranged from 91 to 97% .Non-canonical interactions of the nucleotides result in the diminishing of the thermodynamic stabilities of the duplexes due to the type of mismatches formed. Some single mismatches, such as G-U, G-A, G-G, or U-U, reduce duplex stabilities insignificantly, whereas A-A, C-C or U-C often cause significant changes to their thermodynamic stabilities –30. The ΔΔG°37) between the most stable duplexes formed in the mixture containing WT RNA, Mut RNA, gapmer, and inhibitor. These differences, obtained for all the analyzed RNA SNP types, confirmed the above assumption and were collected in The main assumption of the presented approach was to differentiate the interactions of two types of oligonucleotides with WT and Mut RNA using an appropriate arrangement of 2'-O-methyl and LNA modified nucleotides within the tandem antisense oligonucleotides. As the distinction between canonical and non-canonical RNA-LNA base pairs is much stronger than between RNA-2’OMeRNA, RNA-RNA, or RNA-DNA, especially for internal positions , 34, 35,All the duplexes melted in a two-state manner, indicating that the presence of 2’-O-methylRNA, LNA, and DNA residues do not disturb canonical base pairing , 36. TwoSCA3 mRNA, the better selectivity of Mut RNA degradation was achieved using the shorter inhibitor If1 , but discrimination between duplexes formed by a gapmer (G-T mismatch versus A-T pair) is very poor. In all three analyzed cases, the Kd values obtained for RNA G/A variants of duplexes with a gapmer indicate no significant difference in the binding affinity to particular alleles. This fact confirmed that duplex discrimination between single G-T and A-T base pairs is hardly possible. Also, in these three cases of G/A transition, the SNP sequence context ultimately prejudges the final result. The highest differences in thermodynamic stability between duplexes of WT and Mut RNAs with both oligonucleotides were obtained for the G46A transition within SNCA mRNA, for which the application of the shorter inhibitor better discriminated between the degradation of RNA variants in vitro with RNase H and in HeLa cells , but only 107 nM for Id1/WT 717G RNA (G/A transition). In the case of C/G transversion, it was shown that the presence of the shorter inhibitor Ib1 in the reaction mixture together with the Mut RNA has no influence on RNA degradation because the gapmer bKM forms a more thermodynamically stable (ΔG°37) duplex with the mutated RNA  than the competing Ib1/Mut RNA duplex . The opposite situation was observed for WT RNA, where, at 0.75 μM of Ib1, the inhibition of WT RNA cleavage increased significantly, reaching over 50%. With increasing concentrations of Ib1, a slow gradual inhibition of WT RNA degradation was observed. The maximum inhibition of WT RNA cleavage (78%) was reached at 20 μM of Ib1. The thermodynamic stabilities of Ib1/WT RNA  and bKM/WT RNA  duplexes are fairly similar, and thus the gapmer bKM can also bind WT RNA at low concentrations of Ib1. When the amount of the inhibitor Ib1 increased, most of the bKM/WT RNA duplex was converted into the Ib1/WT RNA duplex and the degradation of the WT RNA was limited. Based on studies performed with 1 μM of inhibitor, a final inhibitor concentration of ten-fold of the target RNA and one hundred-fold of the gapmer was selected. It was shown , three , and four  oligonucleotides indicated the increasing potential for allele-selective RNA degradation . The anaHeLa transfection experiments were performed with each of the antisense oligonucleotides separately at concentrations of up to 100 nM. In the case of C/G transversion, the results obtained for transfection with inhibitors showed a significant influence on the expression of GFP for both alleles  did not result in a decrease of WT RNA cleavage, whereas the presence of the longer inhibitor (Ie2) diminished cleavage by 80% and 40% for WT and Mut RNAs, respectively (HeLa cells with the gapmer and shorter inhibitor (Ie1) significantly decreased the expression of Mut RNA and WT RNA, but the presence of the longer inhibitor (Ie2) resulted in an expression selectivity of 45% in favor of WT RNA. The results from RNase H and HeLa cell assays differed more between each other when the shorter inhibitor was applied, which could suggest an increased specifity of Ie2.A substitution in the contiguous codon 693 of the ectively . CotransHeLa cells, however, no selective silencing was obtained for any of the inhibitors  mRNA , no statistically significant selective silencing of the mutated RNA was observed, whereas the difference in the level of expression between WT and Mut RNA was only 27% for the longer inhibitor (Ia2).In the case of the C4U transition, the cleavage efficiency of WT RNA in the presence of the shorter inhibitor (Ia1) observed in radation . The lonIn vitro RNase H assay results for G291C SNP within SCA3 mRNA  is often an inefficient target for allele differentiation; therefore, other sequence variations (such as short deletions or SNPs), which only remain in linkage disequilibrium with a particular allele, become a target for this strategy. Herein, almost all target SNPs are mutations directly associated with neurodegeneration resulting from protein structure changes related to the occurrence of SNP in its coding region. However, the differentiation of the allele’s expression level on the basis of a single mismatch occurring within siRNA duplexes or RNA/DNA duplexes of SNP variants of RNA is not as obvious and simple as it might seem.Allele-selective silencing of different genes is described for some neurodegenerative diseases inherited in a dominant manner, including Huntington disease, different types of spinocerebellar ataxias, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis (ALS) –44. For ΔΔG°37) of ASO duplexes ranged within 12.12 and -4.03 kcal/mol. The most optimal situation would be if the inhibitor preferentially bound to the WT RNA (inhibitor/WT RNA) while the gapmer bound to the Mut RNA (gapmer/Mut RNA) and promoted RNase H activity. Analysis of the obtained data also indicated that the largest difference in thermodynamic duplex stabilities occurs when the inhibitor is shorter. On average, the differences in thermodynamic stabilities were 7.3 and 2.4 kcal/mol for duplexes of the short inhibitor/WT RNA and gapmer/Mut RNA and the longer inhibitor/WT RNA and gapmer/Mut RNA duplexes, respectively.Single mismatch was reported as a target for allele discrimination mostly with the use of RNA interference , 55–57. in vitro experiments and by 2- to 15-fold for HeLa cell experiments, with the exception of the RNA carrying the C4U SOD1 SNP. Differentiation in the abilities of the SNP-selective cleavage of WT and Mut RNAs is somehow correlated with the thermodynamic stabilities of the duplexes formed by the inhibitors and gapmers with the WT and Mut RNAs. When the interaction of inhibitors and gapmers with the Mut RNA generated A-C and C-C mismatches,  the process of distinguishing WT and Mut RNA was more efficient. The other types of mismatches did not cause significant differences in RNA variant stability.The results presented above allow some conclusions to be drawn concerning the tandem oligonucleotide approach for SNP-selective RNA cleavage. The results summarized in HeLa cell experiments, the observed selectivity of the tandem approach was less clear. Based on thermodynamic rules, the application of the shorter inhibitor should better differentiate its selective binding to WT and Mut RNAs. This dependency was observed mainly when the inhibitor hybridized to the Mut RNA, generating strongly destabilizing base pairs such as A-C (G/A transition), although it was not a rule for the C-C mismatch (C/G transversion) [in vitro. This result was likely associated with the stronger pairing of the inhibitor with the WT RNA and the lower affinity of the gapmer to the WT RNA in view of the intensely destabilizing A-C and C-A mismatches. The results obtained suggest that SNP sequence context and the type and place of nucleotide modification within the ASO are significant for allele-selective discrimination of pathogenic RNA as, in general, they control the final difference in total duplex stability. This difference is crucial during oligonucleotide competition for hybridization with particular RNA variants. Experiments in vitro containing model WT and Mut RNAs as well as both an inhibitor and a gapmer confirmed that the tandem approach significantly enhanced SNP-selective allele discrimination. HeLa cell experiments showed that the use of the shorter inhibitor resulted in allele-selective discrimination more often than the longer inhibitor. Certain differences in the selectivity of RNA inhibition which were observed between results obtained in vitro and in HeLa cells could be a consequence of the tertiary structure of the target RNA as well as its intermolecular interactions inside the living cell. These types of interactions usually play a minor role in in vitro conditions, which result in less molecular crowding and for which it is easier to determine and predict the behavior of molecules. The Dependency of ASO activity relative to target RNA structure has already been reported [in vitro and in cell culture conditions. In cells, where ASO could interact not only with the designated fragment of target RNA but also with other RNAs present in its vicinity, adverse off-target effects could appear due to complementary and partially mismatched interactions, which was noted for ASOs and siRNAs [HeLa cells as molecules of 100 nucleotides, structure prediction was performed in RNAStructure 5.0, which did not indicate significant barriers for efficient ASO hybridization. For the same molecules analyzed in vitro in the RNase H assay, digestion results were comparable with those obtained for 13-nucleotide RNAs.In the version) . In the reported , 59 and d siRNAs . The scain vitro were noted. In most cases, the simultaneous presence of all cleavage reaction components  significantly enhanced SNP selectivity for RNA cleavage. Moreover, in certain cases, the application of the longer inhibitor results in better allele discrimination. For experiments in HeLa cells, the correlation between the thermodynamic parameters and allele-selective degradation is less obvious. It concerns not only the role of the longer and shorter inhibitors but also the reaction mixture itself, which contains many components that can interact with each other. The thermodynamic rules for nucleic acid folding were not determined for intracellular conditions and the crowding effect in cells. The contingency of other potential interactions can limit the thermodynamic dependence of the SNP-selective cleavage of target RNAs using tandem ASO oligonucleotides.Based on the obtained results, some correlations between the thermodynamic stabilities of RNA duplexes and allele-selective degradation in vitro; however, the observed selectivity was less efficient in HeLa cells. In the case of single substitutions, selectivity is highly dependent on the nature of the SNP and surrounding nucleotides. Significant differences in the thermodynamics of the duplexes formed by the inhibitor and gapmer with the WT and Mut RNAs often improved the SNP-selective degradation of Mut RNA. The tandem ASO approach is not a universal method, but taking into account the SNP type, length and sequence of 2’-O-methyl inhibitor oligonucleotide, as well as the nucleotide composition of the modified gapmer, the tandem ASO approach could be successfully applied to support the allele-selective degradation of some pathogenic RNA variants that differ by one nucleotide.The collective results presented here indicated that the tandem approach enhanced SNP selectivity between two RNA alleles S1 FigMost of the analyzed RNAs were totally degraded up to twenty minutes of RNase H hydrolysis. This could be seen also in the charts presenting RNase H hydrolysis efficiency depending on oligonucleotides content in the reaction mixture Click here for additional data file.S2 FigStandard curves for GFP  β-actin , and their superposition for relative gene expression normalization (lower chart—C). Parameters of the curves were determined with Bio-Rad CFX Manager 3.0 software. As the standard curves for target and reference genes run in parallel, expression changes were determined with direct method based of comparison of the normalized target expression with the control sample, which usually was transfection of plasmid constructs only.(TIF)Click here for additional data file.S3 FigIf2 inhibitor changes the WT RNA migration under denaturing gel conditions (stable duplex in 7M urea).(TIF)Click here for additional data file.S4 Fig(A) Gel image after electrophoresis of G46A alleles cleavage of SNCA RNA with RNase H and Ik1/kKM or Ik2/kKM antisense oligonucleotides. (B) and (C) Fluorescence microscope images of HeLa cells (magnification 10x) after 24h cotransfection with WT/Mut G46A -pEGFP constructs and concentrations of inhibitor  and gapmer antisense oligonucleotides, that give statistically significant difference between both alleles expression.(TIF)Click here for additional data file.S5 Fig(A) model WT RNA, (B) model Mut RNA. Numbers above the path refer to inhibitor concentration (μM), arrows indicate cleavage sites.(TIF)Click here for additional data file.S6 Fig(A) WT 692C RNA and (B) Mutated 692G RNA.(TIF)Click here for additional data file.S7 Fig(B) and (C) Fluorescence microscope images of HeLa cells (magnification 10x) after 24h cotransfection with WT/Mut C692G -pEGFP constructs and concentrations of inhibitor  and gapmer antisense oligonucleotides, that give statistically significant difference between both alleles expression.(TIF)Click here for additional data file.S8 FigStatistically significant difference (P<0.05) are marked by asterisks.(TIF)Click here for additional data file.S9 FigUpper charts present kinetics of RNase H hydrolysis, lower charts—efficiency of hydrolysis depending on presence of reaction mixture components. Statistically significant differences between mean hydrolysis efficiency of WT and Mut RNA cleavage are marked with asterisk (TIF)Click here for additional data file.S10 FigStatistically significant differences between mean normalized GFP expression of analyzed RNA variants are marked with asterisk (P<0.05)(TIF)Click here for additional data file.S11 FigT1—RNase T1 cleavage of 692G RNA, F—formamide hydrolysis of 692G RNA, K—control sample without gapmer oligonucleotide (bKM concentration of 0)(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


The aim of this review is to provide a better understanding of lncRNA biological functions by studying them at the structural level.Although thousands of long non-coding RNAs (lncRNAs) have been discovered in eukaryotes, very few molecular mechanisms have been characterized due to an insufficient understanding of lncRNA structure. Therefore, investigations of lncRNA structure and subsequent elucidation of the regulatory mechanisms are urgently needed. However, since lncRNA are high molecular weight molecules, which makes their crystallization difficult, obtaining information about their structure is extremely challenging, and the structures of only several lncRNAs have been determined so far. Here, we review the structure–function relationships of the widely studied lncRNAs found in the animal and plant kingdoms, focusing on the principles and applications of both  Two types of RNA molecules exist : messengIt is generally believed that lncRNAs, RNA molecules longer than 200 nucleotides, belong to a group of RNAs with broad biogenesis, and that these molecules are always capped and polyadenylated . InitialInitially, methods such as nuclear magnetic resonance (NMR) and X-ray crystallography were used for the investigations of RNA structures . HoweverlncRNAs, which are frequently involved in transcriptional, post-transcriptional, and epigenetic processes, are currently the focus of genetic research ,17. PrevXist (X-inactive specific transcript) gene plays an essential role in XCI. The lncRNA Xist, 17 kb in length, is a transcript of Xist, which initiates XCI by coating the X chromosome in order to regulate cis X inactivation (Xi), and by recruiting modifying complexes, such as polycomb repressive 2 (PRC2), to specific sites on Xi, resulting in histone H3 lysine K27 trimethylation (H3K27me3) and X-linked gene silencing . Al. Al28]. et al. confirmed the significance of Xist D-repeat in XCI using CRISPR/Cas9 -associated endonuclease 9) . Co. Co88]. ING-MaP) , developP domain . AlthougP domain . DimethyP domain ; therefoet al., is a novel strategy for genome-wide analysis of RNA structures , suggesting that the less structured UTRs may expose functional elements, while the highly structured coding regions tend to be protected from conformational changes and have the potential to regulate ribosome translocation . Th. Thet allocation . Even thncordant . PARS isncordant , and it Another nuclease-based approach is fragmentation sequencing (FragSeq), which utilizes P1 endonuclease to digest single-stranded RNA, followed by high-throughput sequencing and bioinformatic analyses of the generated fragments  95]. Al. Al95]. et al. determined the functional significance of base-paired RNAs in A. thaliana using dsRNA-seq. They found that the exons of A. thaliana genome enriched with many base pairings were significantly less evolutionarily conserved than other regions, such as 3’UTRs, 5’UTRs, and introns, suggesting that base-pairing interactions were disfavored in the protein-coding regions of plant mRNAs . ss. ss90]. nt mRNAs . In addierase 6) . Even therase 6) . To date elegans ,98,99.in vitro can provide information about RNA secondary structure, but in vitro results cannot be completely extrapolated to in vivo conditions. Therefore, in vivo methods for RNA structure probing are urgently needed in order to decode RNA structure–function relationships. Currently, different chemical reagents that are able to penetrate rapidly into all cellular compartments are used in these in vivo methods. DMS, which directly methylates the base-paring faces of A and C of RNA in loops, bugles, mismatches, and joining regions, is the first RNA structure probing reagent used in living cells . T. T109]. et al. applied hiCLIP in the studies of duplex structures bound by a dsRBP, termed Staufen 1 (STAU1), which is involved in mRNA localization, stability, and translation. The results showed that almost 70% of duplexes can be found in 3’ UTR and duplexes in CDS tend to have shorter loops than in the UTRs . C. C111]. et al. developed a more general method, based on the principles similar to the principles of CLASH and hiCLIP, called RPL (RNA Proximity Ligation) , U1 spliceosome RNA (snR19), and U2 spliceosomal RNA homolog (LSR1) in yeast and human cells [Ramani igation)  112]. I. Iet al.an cells . Additioin vitro, but lncRNA structures in vivo, which are less structured and more dynamic, can often differ dramatically from the in vitro structures. One possible reason for this is that some reagents lack the ability to penetrate cells, which severely limits their usage in vivo [in vivo, which may inhibit or affect the interactions between these reagents and target lncRNAs. Therefore, the development of new methods that can solve the currently existing problems is urgently required. An increasing number of identified lncRNA conserved structures will provide an improved understanding of lncRNA biological functions.lncRNAs play significant roles during transcription, post-transcription, and epigenetic processes in living cells ,113,114. in vivo ,100,105.


In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity.The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3′ –CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3′–CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability.The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions. The synthesis of proteins and their folding into proper three dimensional structures is of fundamental importance for the maintenance of a functional cellular proteome. The ribosome is the cellular translation machine that can synthesize proteins using messenger RNA as the template and aminoacyl-transfer RNAs as substrates Leishmenia donovaniSaccharomyces cerevisiae and trigger factor in Escherichia coli are reported to act as the first line of defense against protein aggregation of nascent translating polypeptide chains Several studies have demonstrated that the ribosome itself is capable of assisting in folding of proteins spanning a wide range of folds and functions in vitro transcription also has chaperoning ability and is referred to as bDV RNA in this study. Studies on the mechanisms of chaperoning activity of bDV RNA showed that it is a two step process involving its two sub-domains RNA1 and RNA2. The initial binding of the unfolded proteins take place with the RNA1 sub-domain  followed by release of the proteins in their folding competent state by the RNA2 sub-domain (that consists of the remaining stem and loop structure of the RNA domain) E. coli PTC interact with a diverse set of folding proteins Saccharomyces cerevisiaeSurprisingly, despite of the large number of proteins associated with this ribonucleoprotein complex, like the peptidyl transferase ability, the chaperoning activity of the ribosome also originates in its ribosomal RNA. Both the peptidyl transferase activity and the chapernoning ability of the bacterial ribosome resides in the domainV of 23S rRNA of the bacterial large ribosomal subunit 3′- CCA ends of A- and P-site tRNAs respectively, have provided important insights into the mechanism of peptidyl transferase reaction in vitro studies presented here explore the effect of tRNAs and antibiotics on the chaperoning ability of the E. coli ribosome and its bDV RNA using bovine carbonic anhydrase (BCAII) as the substrate protein. We demonstrate using mRNA bound 70S ribosomes (programmed ribosome) that a tRNA positioned at the P-site is more effective than an A-site tRNA in inhibiting ribosome's chaperoning function. We also show that the antibiotic blasticidin, a P-site substrate analog The mechanism by which the PTC executes its peptidyl transferase activity has been extensively studied. The ribosome has structurally distinct aminoacyl (A), peptidyl (P) and exit (E) sites that are rich in conserved RNA elements which stabilize tRNA and mRNA substrates in the structurally characterized ‘classical’(C) position via specific interactions. Classically bound L-shaped tRNA molecules orient perpendicular to the subunit interface such that their 3′-aminoacylated – CCA ends projects into the 50S subunit PTC, while the tRNA anticodon pair with mRNA codons within the small ribosomal subunit's decoding region. The 3′-CCA ends of the peptidyl- and aminocyl-tRNAs at the P-site and A-site are the acceptor and donor substrates of the peptidyl transferase reaction respectively. Their accurate positioning via interactions with specific nucleotides of the PTC is crucial for the peptidyl transferase reaction E. coli MRE 600 cells in vitro transcribed domain V of E. coli 23S rRNA (625 bp) is referred to as bDV RNA in this work. The DNA corresponding to this domain, cloned in plasmid pTZ57R/T, was a kind gift from laboratory of Professor C. Dasgupta . The cloned DNA was linearized using EcoRI restriction enzyme (Fermentas) and the RNA was synthesized by run-off transcription using T7 RNA polymerase. The template DNA was removed with RNase free DNase (Fermentas), extracted using phenol-chloroform and the RNA was precipitated using ethanol.Ribosomes were purified from The denaturation and refolding of the BCAII protein either in presence or in absence of the ribosome or bDV RNA were performed under conditions reported in literature E. coli ribosome, b) the bDV RNA, c) ribosome–antibiotic complexes and d) ribosomes (mRNA programmed/non-programmed) complexed to total E. coli tRNA (Sigma Aldrich) or Met-tRNA or Glu-tRNA. As stated in in vitro studies on protein synthesis are carried out in buffers containing K+ as the positive cation. Replacement of NaCl with KCl in the refolding buffer did not affect reactivation yield both during assisted and unassisted refolding of BCAII.The chaperone and its complexes used in this study are, a) the in vitro transcription following which the template DNA was digested with RNase free DNase. The RNA was precipitated with ethanol, centrifuged and resuspended in DEPC treated water and the concentration of mRNA determined by measurement of optical density at 260 nm.Double stranded oligonucleotides (synthesized from Sigma Aldrich) were designed  and the growth condition and the purification procedure were followed E. coli DH5α containing the overproducing plasmid pKR15 was grown at 37°C under aerobic conditions in M9 minimal medium with 11 mM dextrose and 15 mM thiamine, harvested at the end of the exponential phase and resuspended in 20 mM Tris-HCl, pH 7.2. RNA was extracted with phenol, precipitated from the aqueous phase with 2 vol. of ethanol and 0.1 vol. of potassium acetate 20% pH 6.0, and dissolved in 1M NaCl. After centrifugation at 8000 g during 40 min, the clear RNA solution was diluted fivefold with water and loaded on a column of Q Sepharose (Pharmacia) which was then washed with 200 mM NaCl  before eluting the tRNA with 1.5 M NaCl . This unfractionated tRNA was deacylated by incubation at 37°C for 20 minutes in 0.5 M Tris-HCl, pH 8.9. Purification of the glutamate accepting tRNAs from 1000 A260 units of tRNA was performed by chromatography on a 21.2 mm×250 mm column of C18 ODS-Hypersyl (Shandon) coated with trioctylammonium chloride, using a linear salt gradient made with 0.5 M CH3COONH4 pH 4.5, 0.2 mM dithiothreitol, 1 mM EDTA and 5 M CH3COONH4 pH 6.0, 0.2 mM dithiothreitol, 1 mM EDTA. Each glutamate accepting tRNA was purified to homogeneity by 2 or 3 successive passages on this column . Samples were concentrated with a Q-Sepharose FF column (Pharmacia) and desalted using Centricon-10 devices (Amicon). Purified Glu-tRNA was quantified and the accepting capacity of Glu-tRNA was measured A260 units.It has been reported that in solutions of near-neutral pH E. coli tRNA, deacylated Met-tRNA or Glu-tRNA to the ribosome were performed in Buffer-P or Buffer-A E. coli tRNA bound empty ribosomes were prepared by incubating 70S ribosome (0.25 µM) with total E. coli tRNA  was incubated with 3.75 mM tetracycline, total E. coli tRNA (5 µM) or Met-tRNA (5 µM) in 297 µl of Tet-Buffer To study the effect of blocked A-site of empty ribosome on tRNA mediated inhibition of chaperoning ability, the ribosome was first allowed to bind to tetracycline and the effect of total in vitro transcribed mRNA, prepared as described above bound to the 70S ribosome is referred to as programmed ribosome in this study. The binding of mRNA and Met-tRNA (P-site) or Glu-tRNA (A-site) to the ribosome were performed in Buffer-P and Buffer-A respectively  with 2 fold molar excess of mRNA and Glu-tRNA  can also assist in protein folding albeit to a lower extent than that compared to the ribosome in vitro transcribed bDV RNA E. coli tRNA, Met-tRNA and antibiotics using the specific buffer conditions necessary for their binding  and RNA1b  using XbaI (Fermentas) restriction site. Mutations that were introduced in DNA corresponding to bDV RNA, RNA1 and RNA2 were confirmed by sequencing. The RNA was synthesized by run-off transcription as described earlier.Mutations U2585C (in the RNA1 region) and delG2252 (in the RNA2 region) were available in the laboratory. Mutations delU2585, G2252C, A2602C, delA2602, A2451G, delA2451, G2553C were introduced in appropriate positions of DNA corresponding to bDV RNA, bDV RNA1 and bDV RNA2. These mutants were generated using appropriate primers and site directed mutagenesis kit (Stratagene). The mutations were confirmed by sequencing.32P] labelled RNA1 were incubated with equimolar concentration of refolding BCAII for different time intervals, filtered through 0.22 µM pre-soaked nitrocellulose filter paper (Millipore), and 32P counts retained on the filter measured by liquid scintillation counter (Perkin Elmer). Comparing the radioactive count incorporated in the total RNA to that on the filter, the percentage of radioactivity retained was calculated. To follow the RNA2 mediated release of RNA1 from the RNA1-protein complex, the unfolded protein was allowed to form a stable complex with the [α- 32P] labelled wt RNA1 for 5 minutes. Mutant or wt unlabeled RNA2 was then added to the reaction mixture at the same concentration as RNA1. Aliquots withdrawn at different time intervals were filtered through nitrocellulose filter paper and radioactivity retained on filter paper measured. In all these studies the wt counterpart of RNA1 and RNA2 served as the control. In filter binding studies with bDV RNA or its mutants, the radiolabelled RNA was incubated with refolding BCAII. The samples were withdrawn at different time intervals, cross-linked at 254 nm for 90 seconds , filtered through nitrocellulose filter paper and the radioactivity retained on the filter were determined.Filter binding studies were performed to compare binding and release of the refolding protein with wild type (wt) and mutant RNA1 and RNA2 using the procedure as reported earlier in vitro transcribed RNAs (0.1 µM) were dissolved in 20 mM sodium phosphate buffer (pH 7.6) and the CD spectra were recorded from 200 to 300 nm using 1 cm quartz cuvette in a Jasco J-815 CD spectrometer.Circular dichroism spectroscopy was performed with bDV RNA and its mutants (M1-M9). For CD measurements the in vitro transcribed tRNA bound to bDV RNA under buffer conditions conducive to P-site binding had indicated that the P-site tRNA inhibits the protein folding ability of this RNA domain  and Met-tRNA (5 µM) on folding ability of tetracycline bound ribosome was then tested. Appropriate control experiments of tRNA mediated inhibition of ribosome's chaperoning ability in the same buffer were also performed. The inhibition observed in presence of both tRNAs remains essentially unchanged  and GAA (Glu) codons, 7 bp downstream to a ribosome binding site . Since, the Shine-Dalgarno (SD) sequence increases the affinity for mRNA binding to the ribosome by an order of magnitude E. coli tRNA. This might be expected only if the mRNA programming of the ribosome had led to increased specificity of the P-site for Met-tRNA, which represents only a subpopulation of the total E. coli tRNA. Earlier studies have also demonstrated that while in absence of mRNA, the P-site tRNA establishes contacts almost exclusively with the 50S subunit, additional contacts between the tRNA and the 30S subunit is established upon codon-anticodon interaction in presence of mRNA In the absence of mRNA, comparable Met-tRNA mediated inhibition of ribosome's chaperoning ability was achieved only at a concentrations approximately four fold higher  than thaNext, the ribosome was bound to Met-tRNA at a concentration at which partial inhibition of the ribosome's chaperoning ability was observed   . PuromycThe macrolide antibiotics block the polypeptide exit tunnel of the ribosome and thus inhibit extrusion of the nascent chain dOur studies with erythromycin and josamycin show that both these antibiotics can inhibit ribosome mediated protein folding in a dose dependent manner with maximal inhibition achieved at concentrations much higher than their Kin vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) can also assist in protein folding. Studies using this RNA have provided several important insights into the mechanism of the ribosome's chaperoning activity E. coli tRNA, deacylated Met-tRNA and the antibiotics on bDV RNA mediated chaperoning activity were therefore performed. While total E. coli tRNA, deacylated Met-tRNA, blasticidin showed complete inhibition at concentrations indicated in legend of As stated earlier, the in vitro transcribed RNA. This also provides additional support to the fact that the observed inhibition of the chaperoning action with blasticidin or puromycin observed here is not due to any nonspecific effect.Studies on the effect of macrolide antibiotics on domain V RNA mediated chaperoning ability were performed. Neither erythromycin nor josamycin could inhibit the chaperoning activity . The macTaken together, the studies with blasticidin  and puromycin  further establish that the interactions between 3′–CCA end of the tRNA and P-loop and not the A-loop are inhibitory for the chaperoning action of bDV RNA and the ribosome. In this study we also show that the macrolides that target the exit tunnel hydrophobic crevice at the PTC affect chaperoning ability of the ribosome but not of its bDV RNA.in vitro transcription as stated in ‘Based on the studies stated above, we introduced mutations in bDV RNA at sites essential for P-site tRNA binding. Universally conserved nucleotides like G2252, U2585 and A2451 have been shown to be involved in P-site tRNA binding A comparison of the effect of deletion or substitution mutations at these sites on chaperoning ability of bDV RNA is shown in in vitro transcribed domain V rRNAs are structured under the refolding condition. However, most of these mutants showed highly reduced refolding activity, less than 50% compared to the wild type. Mutations in regions corresponding to the protein or 6AP (stated above) interaction sites  showed highest defect in their chaperoning activity. Of the two mutants of bDV RNA that had significantly reduced chaperoning ability in our study, the U2585C mutation lie in proximity to one of the aforementioned RNA-protein interaction sites.Hence, of the nine point mutations studied here, two mutations resulted in a significant reduction in chaperoning activity of bDV RNA. In this context, recently reported studies on refolding ability of mutated variants of domain V of 23S rRNA requires mention 32P] labelled RNA would be retained on the filter paper only if bound to the refolding protein, while the unbound RNA passes through it. The time course of interaction between RNA1 (mutant or wild type) and the refolding protein and its subsequent RNA2 (mutant or wild type) mediated release would therefore be reflected by the amount of radioactivity retained on the filter.As stated earlier the bDV RNA mediated chaperoning is a two step process in which the RNA1 sub-domain initially binds the refolding protein and the RNA2 sub-domain releases the bound protein in a folding competent state. As reported earlier, filter binding studies with wild type or mutant RNA1 and RNA2 have provided insights into the underlying mechanism of loss of chaperoning activity of the bDV RNA mutants. Mutations were introduced in the DNA corresponding to RNA1  and RNA2  as described in “As shown in Parallel ongoing experiments in our laboratory on the effect of bDV RNA on aggregation and refolding of molten globule form of BCAII had also shown that the delG2252 and U2585C mutant could efficiently suppress BCAII aggregation due to their inability to release the bound protein , PLoS One. 9(5): e96425). In filter binding studies with wt bDV RNA and its mutant bDV RNAdelG2252, a lack of release of the protein is observed with the mutant RNA . While BThe role of the ribosome as the cellular protein synthesis machine is well established. The ability of the ribosome to assist in refolding of proteins in vivo studies using PSI+ and Ure 3 strains of yeast Saccharomyces cerevisiae have identified the ribosomal RNA as target for two antiprion drugs, 6AP and GA Saccharomyces cerevisiae imply the presence of a pool of non-translating ribosomes under normal cellular conditions The discussion of some recent observations are relevant in context of the above studies that imply that actively translating ribosomes with a tRNA positioned at the P-site would be unable to perform their chaperoning function. Firstly, the question as to whether the ribosome acts as a chaperone by trans (that is on other proteins) or cis (that is on the same polypeptide that it has synthesized) mechanism still remains to be answered. In the cis-mechanism, the conflict of the two concepts, the ribosome exit tunnel and protein folding on the PTC of the 50S subunit arises where indeed the question of how the nascent protein associates with the PTC after being released from the exit site of the tunnel needs to be further resolved Figure S1A) Control experiments were performed to test the effect of tRNAs and antibiotics on BCAII reactivation in absence of chaperone. Bar diagram shows the effect of total E. coli tRNA (2), Met-tRNA (3), blasticidin (4), puromycin (5), erythromycin (6) and josamycin (7) on the self refolding of BCAII (1). The concentrations of all the tRNAs and antibiotics were the same at which their maximum inhibition of protein folding activity was observed with ribosome . (B) Comparison of BCAII reactivation after 30 minutes of refolding in absence of chaperone (1), in presence of 10 µM heparin (2), in presence of 70S ribosome (3), and in presence of 70S ribosome +10 µM heparin (4).Effect of antibiotics, tRNA and heparin on BCAII reactivation. ((TIF)Click here for additional data file.Figure S2A) Wild type (wt) bDV RNA and its nine mutant variants were subjected to CD analysis to assess the secondary structure. The measurements were done in phosphate buffer (20 mM) using a quartz cuvette with a path length of 1 cm in a Jasco J-815 CD spectrometer with an RNA concentration of 0.1 µM. (B) Filter binding studies on the time course of interaction between BCAII and wt bDV RNA (•), delG2252bDV RNA (▪) are shown here. (C) Comparison of the BCAII reactivation after 30 minutes of refolding in absence of chaperone (1), and in presence of wt bDV RNA (2), delG2252bDV RNA (3), delG2252bDV RNA + wt RNA2 (4).Effect of mutations of bDV RNA on CD spectra and its effect on the binding-release and reactivation of BCAII. ((TIF)Click here for additional data file.Table S1Summary of buffer systems used in this study.(DOC)Click here for additional data file.Table S2Summary of dissociation constants of antibiotics.(DOC)Click here for additional data file.


The new landscape of human transcriptome along with the identification of numerous long non-coding RNAs (lncRNAs) has dramatically altered our approach to study diseases. It is now imperative to decipher the biological functions of these transcripts and how they impact on human cell and pathophysiology. Nonetheless, already at this very early stage of their study, the involvement of lncRNAs in cell transformation is emerging as a key aspect. Recently, researchers have started to explore the implications of lncRNAs alteration in hepatic pathophysiology. In this review, we will discuss in detail several examples of liver disease-relevant lncRNAs. Many lncRNAs have been shown to play a major role in hepatocellular carcinoma (HCC). For such type of tumor with an increasing incidence and a high mortality rate, it is crucial to identify new therapeutic targets and biomarkers to predict response to therapy. LncRNAs present as a promising new resource. One major challenge for the future would be to systematically address the lncRNAs expression among the different cellular components of the liver. To achieve this goal, a combination of clinically driven, genetically defined, morphologically classified, and molecular-based studies will have to be performed. In conclusion, lncRNAs will undoubtedly provide a rewarding field of study and most importantly a new resource to identify new disease associated biomarkers and molecular targets for therapy for liver diseases. In addinse RNA) , 15, havnse RNA) . Howevernse RNA) , 17. Recnse RNA) . We willnse RNA) . At the nse RNA) , thus reHepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide and represents the majority of liver cancers  , 21. HCC-β), and PTEN/Akt  experiments. LALR-1 facilitates cyclin D1 expression through the activation of Wnt/β-catenin signaling via the suppression of Axin1  and ABCA1 expression has been shown to modulate both glucose and cholesterol metabolism in liver, eventually resulting in reduced atherosclerotic plaque progression in apoE(−/−) mice  mice . These rLong non-coding RNAs are increasingly recognized as essential regulators of biological processes in normal and pathological conditions thus having a high potential to serve as novel diagnostic and prognostic tools . Many inThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The G-quadruplex (G4) is a non-canonical nucleic acid structure which regulates important cellular processes. RNA G4s have recently been shown to exist in human cells and be biologically significant. Described herein is a new approach to detect and map RNA G4s in cellular transcripts. This method exploits the specific control of RNA G4–cation and RNA G4–ligand interactions during reverse transcription, by using a selective reverse transcriptase to monitor RNA G4-mediated reverse transcriptase stalling (RTS) events. Importantly, a ligation-amplification strategy is coupled with RTS, and enables detection and mapping of G4s in important, low-abundance cellular RNAs. Strong evidence is provided for G4 formation in full-length cellular human telomerase RNA, offering important insights into its cellular function. G-quadruplex G4) nucleic-acid structures play pivotal roles in the regulation of a myriad of cellular processes, and have nucleic-+  can be rationally controlled in a cation- and G4 ligand-dependent fashion Figure . NotablyNRAS,/Li+), and 5-fold (Li+[PDS]/Li+) RTS effect for NRAS G4 ,hTERC), a 451 nt long noncoding RNA (lncRNA) critical for the regulation of telomere length., about 150 mM. We cannot rule out the possibility that both structures could form simultaneously with a quadruplex comprising a C bulge (nt 1–17).[hTERC,[hTERC needed for telomere maintenance.[hTERC support the fact that enzymes which can resolve G4 in RNA, such as DHX36,[hTERC RNA conformation for telomerase function.The phylogenetic RNA secondary structure of x Figure , a key e00 mM K+. The addi00 mM K+. Combinednt 1–17). Recent p).[hTERC, and thatas DHX36, may playhTERC. Moreover, we show that the approach is applicable to detect and validate RNA targets for G4 ligands. In the future, we will adapt the approach described here to map G4s throughout the transcriptome.In summary, we introduce the RTS-HBLMPCR approach for probing G4 formation and location in full-length low-abundance cellular RNAs. We exemplify the approach by detecting and mapping G4 formation in the biologically important cellular lncRNA,


Stress granules (SG) are formed as collections of protein and RNA (ribonucleoprotein structures) and continuously assembled/disassembled in response to stresses such as heat, osmotic, or oxidant stress; representing an attempt to survive the stress through salvage of important proteins and RNA. Recent research suggests diabetic nephropathy (DN) may change or alter SG biology and in conditions that model DN may involve the receptor for activated C-kinases (RACK1). The incorporation of RACK1 into stress granules may down-regulate programmed cell death and further may impart the ability to scaffold to and sequester key signaling proteins to affect cell survival or death. We hypothesized that the inappropriate or dysregulated scaffolding of proteins or RNA into stress granules may be of importance in the development of diabetic nephropathy. The goal of this study is to qualitatively and semi-quantitatively characterize the effects of cell culture conditions modeling diabetes and ER stress in conjunction with over-expression studies of SG stabilizing proteins on RNA transcripts.Immortalized human proximal tubule cells (HK2) were grown to 70-80% confluence, transfected with GFP, GFP-RACK1, or GFP-dnRACK1 for 24hr and then treated with 0.5uM thapsigargin (Tg) or vehicle (DMSO) for 30min prior to cell lysis and sample collection.Protein lysate (500ug) was fractionated using Optiprep (Sigma Aldrich) density gradient medium to prepare 1+7 layered fractions, containing 2.5% Optiprep plus the 500ug lysate and 5, 10, 15, 20, 25, 50, and 54% Optiprep, into thick walled UC tubes, loaded into a SW 55 Ti rotor and spun at 40.6K rpm for 3hr at 4C (medium acceleration and no brake). Fractions were collected and used for immunoblot analysis and also transcriptomic analyses.Localization of marker proteins to specific UC fractions was achieved by immunoblot analysis for GFP and RACK1 (transfection markers), TIA-1 or G3BP (stress granule markers), and GADPH.Homo sapiens reference genome (GenBank Assembly ID: GCA_000001405.1) using Tophat (version 2.0.4). Assembly was performed using Cufflinks (version 2.1.1) resulting in genes with significantly differential expression. Functional analysis was then conducted by MetaCore .Total RNA was isolated from UC fractions using TRIzol reagent  and yields quantified using either 260/280 reading using a Nanodrop 1000  or Qubit fluorometric quantification . The quality of isolated RNA per UC fraction was assessed using an Agilent 2100 Bioanalyzer. For RNAseq analysis, the samples were sequenced with a 50 bp single-end run on an Illumina GAIIx system at Cofactor Genomics . Bioinformatics analysis was conducted at the Kentucky Biomedical Research Infrastructure Network (KBRIN) Bioinformatics Core. Reads passed quality control were aligned to Analysis of HK2 cell lysates fractionated using Optiprep density gradient UC demonstrated by immunoblot that cytoplasmic proteins were found in the 2.5% and 5% Optiprep fractions while SG markers (TIA1 or G3BP) and RACK1/dnRACK1 constructs were contained in the 5-15% Optiprep fractions. Total RNA yields ranges from the 10-400ng/uL range with the highest yields found in the 50-54% Optiprep fractions. Qualitative analysis of RNA by the Agilent 2100 Bioanalyzer suggests that the 2.5-15% Optiprep fractions selectively contain non-ribosomal RNA while the 20-54% Optiprep fractions contain largely ribosome RNA. Qualitative comparison of RNAseq data suggests the RNA isolated from the 2.5% Optiprep fraction has more degradation than RNA isolated from 5% or 10% Optiprep fractions. As compared to DMSO (vehicle) treatment, Tg treatment induces greater that 2-fold changes  in the abundances of 1,300-1,500 genes in each transfection condition. These differences include lincRNA, antisense RNA, miRNA, and coding RNA transcripts. The strongest difference for Tg+RACK1 transfections was for an 80+ fold increase in the abundance of the coding RNA for CERCAM . The strongest difference for Tg+dnRACK1 transfections was for a 300+ fold decrease in the abundance of the coding RNA for KRAP (actin interacting protein regulating IP3R-mediated Ca++ release). The strongest difference for Tg+GFP transfections was for a generalized increase in the abundance of the coding RNA for histone H2 and H3 cluster variants. These results suggest that RNA abundance both qualitatively and quantitatively is affected by ER stress and the abundances may be modifiable by the transfection of proteins that can stabilize SG.


Defining the RNA target selectivity of the proteins regulating mRNA metabolism is a key issue in RNA biology. Here we present a novel use of principal component analysis (PCA) to extract the RNA sequence preference of RNA binding proteins. We show that PCA can be used to compare the changes in the nuclear magnetic resonance (NMR) spectrum of a protein upon binding a set of quasi-degenerate RNAs and define the nucleobase specificity. We couple this application of PCA to an automated NMR spectra recording and processing protocol and obtain an unbiased and high-throughput NMR method for the analysis of nucleobase preference in protein–RNA interactions. We test the method on the RNA binding domains of three important regulators of RNA metabolism. Understanding how RNA-binding proteins select target RNA sequences is key to explain specificity in RNA regulation, but is a challenging problem. Many protein regulators recognize a diverse ensemble of cellular RNA targets by the combined action of several RNA binding domains . Recogni1H{15N} correlation nuclear magnetic resonance (NMR) spectroscopy is used to compare the average binding affinity of a protein domain to different sets of quasi-degenerate RNA pools  approach can be used to define the sequence preference of a protein domain. In SIA, NA pools . For eacNA pools . These sNA pools –7. HowevNA pools .1H{15N} correlation spectra), into five principal components with associated scores and loadings. PCA has been used to correlate measured NMR observables with conformational transitions, global structural changes and changes in the protein environment  to analyse similar NMR data in a high-throughput fashion and determine the sequence preference of an RNA binding domain. PCA was developed as a technique to reduce the complexity of multi-variable sets of data to a smaller number of new variables  that capture the majority of the variability existing in the data. It allows for the identification of patterns in the data displaying the data in a way that highlight differences and similarities. In the current work, PCA is employed to recast five variables , each with 290 × 256 data points  was expressed in BL21(DE3) (Novagen) and purified using Strep-Tactin resin (IBA). The tag was removed with GST-tagged Human Rhinovirus 3C protease overnight. The protease was then removed using a disposable GST column . The protein was further purified using size exclusion chromatography on a Superdex 75 (16/60) . NMR samples were prepared in 40 mM NaCl, 20 mM Tris pH 7.5, 0.5 mM TCEP.2PO4 pH 6.1, 0.1% B-mercaptoethanol.His-tagged T-STAR KH domain (residues 50–160) was expressed in Rosetta BL21 DE3 and purified using Ni-NTA agarose (Qiagen) followed by TEV cleavage during overnight dialysis in phosphate buffer at 4°C and further purified by size-exclusion chromatography on a Superdex 75 10/300 . Final samples were in 50 mM NaCl, 20 mM NaH2.His-tagged TUT4 CCHC ZnF3 domain (residues 1354–1382) was expressed in BL21(DE3) cells and purified using Ni-NTA agarose followed by TEV cleavage during overnight dialysis in Tris buffer at 4°C. A second reverse Ni-NTA agarose purification removed the His-tag and protease, and lead to high purity samples. Final samples were in 100 mM NaCl, 10 mM Tris pH 7.4, 0.5 mM TCEP, 10 μM ZnCl2O. RNA concentration was determined by UV spectroscopy.All RNAs pools were purchased from Thermo Scientific (Dharmacon). Pools were de-protected following the manufacturer instructions and re-suspended in HSamples of 25 μM RNA15, 40 μM TSTAR, and 100 μM TUT4 were prepared and RNA pools added as required to obtain measurable shifts and depending on the affinity of each protein for RNA and on the overall spectral quality. Protein–RNA ratios were 1:1 for RNA15, 1:2 for TSTAR and 1:4 for TUT4. NMR experiments were performed using 3 mm NMR tubes with a sample volume of 180 μl.15N-SOFAST-HMQC experiments with 32 scans, 64 increments and a recycle delay of 250 ms  gave adequate resolution and sensitivity and were used throughout. For each RNA position, spectra from a control sample and four pools were processed as a pseudo-3D dataset using NMRPipe and passed as input to pcaNMR, a NMRPipe module which employs the NIPALS algorithm to apply PCA to the series of NMR spectra. This efficient algorithm permits the calculation of all five principal components in seconds. An example processing script is available from the authors.NMR data were acquired using a Bruker Avance III NMR spectrometer operating at 700 MHz and equipped with a 5 mm TCI cryoprobe. Automated sample changing and data acquisition were accomplished using a Samplejet accessory controlled with Topspin 2.1 via IconNMR. Samples were stored at 4°C and then pre-heated at 25°C prior to loading. Locking, tuning, matching and shimming were performed automatically. 2D The method was tested on the RNA Recognition Motif (RRM) domain of the yeast mRNA 3′end processing factor RNA15, a core component of the Cleavage Factor 1A (CF1A) complex ,13. CF1A1H{15N}-SOFAST-HMQC spectrum  . In ordespectrum  for eachAfter acquisition, the data were converted and processed in-batch using an NMRpipe-based pipeline. We then calculated the principal components for the group of spectra that define nucleobase preference in position 1 using the NMRPipe module pcaNMR . The calAn initial analysis of the principal component data revealed that, for all four positions, a clear correlation exists between the loadings on the second principal component and RNA-dependent chemical shift changes, i.e. the spectra where the peaks shift more between free and bound also have a second principal component that is more distant from the reference spectrum—despite this component accounting for only ∼7% of the variance. We did not observe any such correlation for the first, third, fourth and fifth principal components . The first principal component  is presumably dominated by changes in peak intensity—the method as used entails a dilution on addition of the RNA pools. The third, fourth and fifth components contribute <5% each of the remaining variance (Supplementary Table S1). This correlation between the second PC and peak shifts has been noted previously in the analysis of qualitative changes in NMR spectra of proteins recorded during high-throughput screening of ligand binding .We then compared the results of manual and PC analysis for the four positions in the RNA target. The absolute values of the SIA scores and of the PCA scores reported in Figure Neurexin gene . Our analysis again showed a comparable ranking of nucleobase preference in PCA and manual approaches both for the KH and ZnF domain.Next, we extended our analysis and probed the effectiveness of this protocol on two other common RNA binding domains. T-STAR, also referred to as Sam68-like mammalian protein 2 (SLM2) is an important splicing regulator of the xin gene . The proxin gene . TUT4 isxin gene ,22. The xin gene –25. The xin gene  were empxin gene  and on oWe present here a principal component-based approach to compare changes in NMR spectra upon binding of RNA oligonucleotides, used as an alternative to manual SIA analysis to determine the specificity of an RNA binding domain. Coupling PCA to automated sample handling, spectrometer control and processing reduces the time required for data acquisition, processing and analysis by an order of magnitude, from several days to several hours.The use of PCA for the analysis of NMR data is well established. However, the use of PCA as a tool to obtain a semi-quantitative ranking of affinities from the direct analysis of 2D dataset has not been attempted before. A first important question was which, if any, of the principal components in our set of spectra correlates with the shift of resonances in the same NMR spectra. The plots of the first five principal components against the weighted chemical shift average of the peaks used in the manual SIA analysis  showed that a good correlation exists only for the second principal component. Such a correlation between chemical shift changes and the second principal component is consistent with the reported use of the second principal component to screen the binding of small molecular weight compounds to a protein, and we observe it consistently for all positions and in all three domains we have examined . The secOur aim is to implement an analysis strategy that does not require a domain-dependent optimization of the parameters of PC analysis and testing the PCA method on three common RNA binding domains highlights possible difference in the output deriving from the domains different sizes and RNA binding modes. The ZnF domain is significantly smaller than the KH and the RRM domains. The total number of amide resonances in the spectrum of the TUT4 ZnF4 is about one-third of the number of amide resonances in RNA15 RRM, and the number of resonances that shift upon RNA binding is proportionally lower . We report that, although the PCA results recapitulate the manual SIA ones for both domains, the range of the PCA scores is significantly smaller for the ZnF domain than for the KH and RRM ones. Indeed examination of the amount of variance accounted for by each PC suggests that the variance is more evenly distributed over the different components (Supplementary Table S1). This suggests that the empirical relationship between spectral features and principal components has been compromised. This observation may serve as a guide to the range of applicability of the PCA-based analysis. In general, it seems that changes in spectra where only very few peaks change position upon RNA binding are more accurately monitored using manual SIA scoring than PCA analysis.in cell methods (e.g. CLIP and its variants) have identified the ensemble of RNA targets for many different multi-domain regulators of RNA metabolism and localization. However, in many of these proteins multiple domains contribute to recognition and an understanding of target selection requires the full characterization of the sequence preference of each individual domain. The manual SIA method provides the required characterization of nucleobase preference but is laborious and time consuming and not designed for the characterization of a large number of domains. Above we show that PCA and automated NMR can be used to perform an unbiased and high-throughput analysis of RNA-binding data and extends the reach of our analysis to characterize the specificity in a large number of protein–ssRNA interactions. Further, we believe the quantitative use of PCA in directly evaluating protein-ligand binding affinities we present here has the potential to be applied in a broad range of comparative NMR binding assays.New Supplementary Data are available at NAR Online.