Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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3âREADS+, a sensitive and accurate method for 3â end sequencing of polyadenylated RNA

ABSTRACT: Sequencing of the 3â end of poly(A)+ RNA identifies cleavage and polyadenylation sites (pAs) and measures transcript expression. We previously developed a method, 3â region extraction and deep sequencing (3âREADS), to address mispriming issues that often plague 3â end sequencing. Here we report a new version, named 3âREADS+, which has vastly improved accuracy and sensitivity. Using a special locked nucleic acid oligo to capture poly(A)+ RNA and to remove bulk of the poly(A) tail, 3âREADS+ generates RNA fragments with an optimal number of terminal As that balance data quality and detection of genuine pAs. With improved RNA ligation steps for efficiency, the method shows much higher sensitivity (over two orders of magnitude) compared to the previous version. Using 3âREADS+, we have uncovered a sizable fraction of previously overlooked pAs located next to or within a stretch of adenylate residues in human genes, and more accurately assessed the frequency of alternative cleavage and polyadenylation (APA) in HeLa cells (~50%). 3âREADS+ will be a useful tool to accurately study APA and to analyze gene expression by 3â end counting, especially when the amount of input total RNA is limited. Nine 3'READS+ libraries were made with different amounts (100 ng, 200 ng, 400 ng, 1000 ng, 5000 ng, 15000 ng) of input Hela RNA.

ORGANISM(S): Homo sapiens

SUBMITTER: Dinghai Zheng

PROVIDER: E-GEOD-84170 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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3'READS+, a sensitive and accurate method for 3' end sequencing of polyadenylated RNA.

Zheng Dinghai D Liu Xiaochuan X Tian Bin B

RNA (New York, N.Y.) 20160810 10

Sequencing of the 3' end of poly(A)(+) RNA identifies cleavage and polyadenylation sites (pAs) and measures transcript expression. We previously developed a method, 3' region extraction and deep sequencing (3'READS), to address mispriming issues that often plague 3' end sequencing. Here we report a new version, named 3'READS+, which has vastly improved accuracy and sensitivity. Using a special locked nucleic acid oligo to capture poly(A)(+) RNA and to remove the bulk of the poly(A) tail, 3'READS ...[more]

PMID: 27512124

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Project description:Small non-coding RNA biogenesis typically involves cleavage of structured precursors by RNase III-like endonucleases. However, guide RNAs that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5â² triphosphate characteristic of transcription start site and possess U-tail indicative of 3â² processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase and 3â²-5â² DSS1 exonuclease, and three additional subunits. This complex, termed mitochondrial 3â² processome (MPsome), is responsible for primary uridylation of ~800-nt gRNA precursors, their processive degradation to a mature length of 50-60 nt, and secondary U-tail addition. Both strands of gRNA gene are transcribed giving rise to sense and antisense precursors of similar size. Head-to-head hybridization of these transcripts blocks symmetrical 3â²-5â² degradation at the fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5â² extremity of a primary molecule by uridylation-induced, antisense transcription-controlled exonucleolytic degradation. 1. we first sequenced guide RNA precursor (Gel-fractioned total cellular RNA 600-1500nt was used) transcripts from three replicates to study the their tail features and also validate observation of sense/antisense accumulation upon perturbation of pre-processing complex based on few cases. 2. we then sequenced mitochondrial small RNA and built a reference for small RNAs using our custom algorithm. We then took the mitochondrial small RNA data and uncovered the sense/antisense pair. 3. We then usedÂ CLIP-Seq data toÂ investigateÂ in vivo binding sitesÂ and, together with RNA IP-Seq data to understand what determine the relative abundance of sense and antisense pair of the duplex. 4. We used sequenced small mitochondrial RNA in different RNAi experiments (For RNAi experiments, 30-70nt RNA fraction was gel-isolated from total cellular RNAs) to understand which protein will affect the Utail length in mature small mitochondrial RNA.

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3âREADS+, a sensitive and accurate method for 3â end sequencing of polyadenylated RNA

Publications

3'READS+, a sensitive and accurate method for 3' end sequencing of polyadenylated RNA.

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3âREADS+, a sensitive and accurate method for 3â end sequencing of polyadenylated RNA