Analysis of alterative cleavage and polyadenylation by 3′ region extraction and deep sequencing

1] Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School, Newark, New Jersey, USA. [2].
Nature Methods (Impact Factor: 32.07). 12/2012; 10(2). DOI: 10.1038/nmeth.2288
Source: PubMed


Alternative cleavage and polyadenylation (APA) generates diverse mRNA isoforms. We developed 3' region extraction and deep sequencing (3'READS) to address mispriming issues that commonly plague poly(A) site (pA) identification, and we used the method to comprehensively map pAs in the mouse genome. Thorough annotation of gene 3' ends revealed over 5,000 previously overlooked pAs (∼8% of total) flanked by A-rich sequences, underscoring the necessity of using an accurate tool for pA mapping. About 79% of mRNA genes and 66% of long noncoding RNA genes undergo APA, but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Quantitative analysis of APA isoforms by 3'READS indicated that promoter-distal pAs, regardless of intron or exon locations, become more abundant during embryonic development and cell differentiation and that upregulated isoforms have stronger pAs, suggesting global modulation of the 3' end-processing activity in development and differentiation.

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Available from: Bin Tian, Oct 25, 2014
    • "Total RNA was subjected to two rounds of poly(A) selection using oligo-d(T) 25 magnetic beads (New England Biolabs). A single-read cDNA library was prepared following the Illumina TrueSeq small RNA protocol for strand-specific RNA-seq with minor modifications (Hoque et al. 2013). Briefly, poly(A) + RNA was fragmented in an alkaline buffer (NaHCO 3 at pH 9.3) for 2 min at 94°C followed by dephosphorylation with recombinant shrimp alkaline phosphatase (New England Biolabs) and then phosphorylation with T4 polynucleotide kinase (New England Biolabs). "
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    • "Red dots indicate genes for which the longer 3 0 isoform was significantly upregulated in the population under investigation; yellow dots correspond to significant upregulation of the shorter 3 0 isoform. Significance was determined by Fisher's test (P < 0.05), see also Hoque et al (2013) "
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    • "Given the above results implicating RBBP6 in 39 processing, we wondered whether lowering RBBP6 levels in cells would affect APA. To this end, we used 39 region extraction and deep sequencing (39 READS) (Hoque et al. 2013) to detect APA changes following RBBP6 knockdown in MCF-7 cells. (We used MCF-7 cells because, as mentioned above, RBBP6 can interact with p53 and we wanted to carry out the knockdown in the background of a functional p53, which is not the case in HeLa cells.) "
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    ABSTRACT: Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3' processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN ("domain with no name"), that is required for 3' processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3' processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3' untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3' processing, especially of RNAs with AU-rich 3' UTRs.
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