Nm-REP-seq Reveals 2’-O-Methylation Landscape at the 3'-end of Noncoding RNAs with Single-base Resolution
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ABSTRACT: Many non-coding RNAs (ncRNAs), such as piRNAs, miRNAs and siRNAs, contain Nm at the 3'-end nucleotides, protecting the 3’-ends form terminal nucleotide addition or exonucleolytic degradation, but whether other kinds of RNAs contain a 3'-terminal Nm remain unknown. Here, we developed Nm-REP-seq to globally profile the 3'end-Nm sites and we revealed several novel classes of Nm-containing ncRNAs in mice and drosophila. Importantly, we discovered that Nm distribute at the end of miRNA, snoRNA, snRNA, tRNA as well as fragments derived from them. Collectively, our approach precisely redefines the genome-wide distribution of Nm and provides new technologies for functional studies of Nm-mediated gene regulation. Method: We developed Nm-REP-seq to globally profile the 3'-end Nm sites. Results: We discovered that Nm distribute at the end of miRNA, snoRNA, snRNA, tRNA as well as fragments derived from them. Conclusion:Collectively, our approach precisely redefines the genome-wide distribution of Nm and provides new technologies for functional studies of Nm-mediated gene regulation.
Project description:2’-O-methylation (Nm) is one of the most abundant RNA epigenetic modification and plays vital roles in the post-transcriptional regulation of gene expression. Current Nm mapping approaches are normally limited to highly abundant RNAs and have significant technical hurdles in mRNAs or relatively rare non-coding RNAs (ncRNAs). Here, we developed a new method for enriching Nm sites by using RNA exoribonuclease (M. genitalium RNase R, MgR) and periodate oxidation reactivity to eliminate 2’-hydroxylated (2’-OH) nucleosides, coupled with sequencing (Nm-REP-seq). We revealed several novel classes of Nm-containing ncRNAs as well as mRNAs in humans, mice, and drosophila. We found that some novel Nm sites are present at fixed positions in different tRNAs and are potential substrates of fibrillarin (FBL) methyltransferase mediated by snoRNAs. Importantly, we discovered, for the first time, that Nm located at the 3’-end of various types of ncRNAs and fragments derived from them. Our approach precisely redefines the genome-wide distribution of Nm and provides new technologies for functional studies of Nm-mediated gene regulation.
Project description:Small RNAs were deep sequenced from the liver and spleen of adult mice in an effort to identify somatic piRNAs. Following sequencing of all small RNAs, known non-coding RNAs were computationally removed from the dataset. The remaining RNAs were then mapped to the genome and analyzed for sequence characteristics (5' base, length) typical of known piRNAs. To determine if any of the identified small RNAs were MIWI2 dependent, we deep sequenced small RNAs from liver and spleen of MIWI2 KO mice and analyzed them as above. We deep sequenced small RNAs from the liver and spleen of one WT mouse and one MIWI2 knock-out mouse. We then trimmed sequencing adapters and removed known ncRNAs (rRNA, tRNA, snoRNA, snRNA, miRNA) from the dataset before aligning reads to the mm9 assembly of the mouse genome.
Project description:Mediator is a co-regulator of RNA polymerase II (Pol II), transducing signals from regulatory elements and transcription factors to the general transcription machinery at the promoter. We here demonstrate that Med20 influences ribosomal protein expression in fission yeast. In addition, loss of Med20 leads to an accumulation of aberrant readthrough tRNA transcripts. The aberrant transcripts are polyadenylated and targeted for degradation by the exosome. Â Similarly, other specialized RNA molecules, such as snRNA, snoRNA and rRNA are also accumulated in the absence of Med20. We suggest that fission yeast Mediator takes part in a regulatory pathway of Pol III transcripts. RNA-sequencing of wt, med20â, rrp6â, med20â/rrp6â strains using wt as control. Each strain was sequenced using three biological replicas. The results were analyzed for differential expression.
Project description:Argonaute/Piwi proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18-22 nt tRNA 3M-bM-^@M-^Y fragments are biochemically distinct from the tRNA halves produced transiently in response to stress. Notably, the end positions of Twi12-bound tRNA 3' fragments precisely match RNAs detected in total small RNA of mouse embryonic stem cells and human cancer cells. Our studies demonstrate unanticipated evolutionary conservation of mature tRNA processing to tRNA-fragment small RNAs. Two libraries are analyzed here: sRNAs associated with slightly overexpressed ZZ-tagged Twi12 purified under native conditions (size range 15-34nt), and those associated after formaldehyde crosslinking (15-22nt).
Project description:Argonaute/Piwi proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18-22 nt tRNA 3’ fragments are biochemically distinct from the tRNA halves produced transiently in response to stress. Notably, the end positions of Twi12-bound tRNA 3' fragments precisely match RNAs detected in total small RNA of mouse embryonic stem cells and human cancer cells. Our studies demonstrate unanticipated evolutionary conservation of mature tRNA processing to tRNA-fragment small RNAs.
Project description:We developed a general method based on RNA Antisense Purification (RAP) to identify the intermolecular RNA-RNA interactions of a target RNA (RAP-RNA). RAP-RNA identifies endogenous RNA-RNA complexes through in vivo crosslinking, RNA capture with antisense oligonucleotides, and high-throughput RNA sequencing. This approach provides a systematic view of other RNAs that interact with a target RNA, and furthermore can distinguish between direct and indirect RNA-RNA interactions through the use of crosslinking reagents with different reactivities with proteins and nucleic acids. We applied this method to numerous small and large noncoding RNAs, including U1 snRNA, Malat1 lncRNA, Xist lncRNA, U3 snoRNA, U17/Snora73a snoRNA and U12 snRNA. We examined the RNA and chromatin interactions of ncRNAs in mouse embryonic stem cells. We developed and applied three related protocols: RAP-RNA[AMT], RAP-RNA[FA], and RAP-RNA[FA-DSG]. In the RAP-RNA[AMT] protocol, we fixed direct RNA-RNA hybrids in mouse embryonic stem (ES) cells with 4'-aminomethyltrioxalen (AMT), a psoralen-derivative crosslinker; AMT generates inter-strand crosslinks between uridine bases in RNA but does not react with proteins. In the RAP-RNA[FA] protocol, we used a different crosslinking strategy to capture both direct and indirect RNA-RNA interactions: we fixed ES cells using formaldehyde (FA), which crosslinks protein-RNA and protein-protein interactions and thus can capture both indirect interactions as well as direct interactions that are caged or flanked by proteins. In the RAP-RNA[FA-DSG] protocol, we fixed with both FA and disuccinimidyl glutarate (DSG), a strong protein-protein crosslinker, to more efficiently capture RNAs linked indirectly through multiple protein intermediates.
Project description:Populations of small eukaryotic RNAs, in addition to relatively well recognized molecules (such as miRNAs or siRNAs), also contain fragments derived from all classes of constitutively expressed non-coding RNAs. It has been recently demonstrated that the formation and accumulation of RNA fragments (RFs) is cell-/tissue-specific and depends on internal and external stimuli. Unfortunately, the mechanisms underlying RF biogenesis and function remain unclear. To better understand them, we employed RNA pull-down and mass spectrometry methods to characterize the interaction networks of seven RFs originating from tRNA, snoRNA and snRNA. In addition, we performed an in silico screen of the selected RFs against publicly available cross-linking and immunoprecipitation datasets. We determined that the RF interactome comprises a large number of proteins, which were generally different from those that interact with their parental full length RNAs. Proteins that were differentially bound by the RFs were involved in mRNA splicing, tRNA processing, DNA recombination/replication, protein biosynthesis and carbocyclic acid metabolism. Our data suggest that RFs can be endogenous aptamer-like molecules and potential players in emerging RNA-protein regulatory networks.
Project description:In this study, we employed a special size fractionation and cDNA library construction method followed by 454 deep sequencing to systematically profile rice intermediate-size ncRNAs. Our analysis resulted in the identification of 1349 ncRNAs in total, including 754 novel ncRNAs of an unknown functional category. Chromosome distribution of all identified ncRNAs showed no strand bias, and displayed a pattern similar to that observed in protein-coding genes with few chromosome dependencies. More than half of the ncRNAs were centered around the plus-strand of the 5’ and 3’ termini of the coding regions. The majority of the novel ncRNAs were rice specific, while 78% of the small nucleolar RNAs (snoRNAs) were conserved. Tandem duplication drove the expansion of over half of the snoRNA gene families. Furthermore, 90% of the snoRNA candidates were shown to produce small RNAs between 20-30 nt, 80% of which were associated with ARGONAUT proteins generally, and AGO1b in particular. Overall, our findings provide a comprehensive view of an intermediate-size non-coding transcriptome in a monocot species, which will serve as a useful platform for an in-depth analysis of ncRNA functions. Examination of non-coding RNA in 2 stages in Oryza sativa, using 454 deep sequecing
Project description:Smal RNA is a type of single-stranded small-molecule RNA with a size of about 18-40 bases, mainly including microRNA, piRNA, snoRNA, snRNA, tRNA and so on. Small RNAs have important regulatory functions in cells and have the potential to be used as disease diagnostic markers or drug targets. We report the results of all small RNAs in exosomes from HTNV infected/uninfected HUVECs by high-throughput sequencing technology. We find that the transcriptomes of Exo-NC group (exosomes from HTNV uninfected cells) and Exo-HV group (exosomes from HTNV infected cells) expressed distinctly different expression patterns of miRNA.