ARTICLE DOI: 10.1038/s41467-017-02099-7 OPEN Post-transcriptional 3´-UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments Yuval Malka1, Avital Steiman-Shimony2, Eran Rosenthal 3, Liron Argaman2, Leonor Cohen-Daniel1, Eliran Arbib1, Hanah Margalit 2, Tommy Kaplan 3 & Michael Berger 1 1234567890 The majority of mammalian genes contain one or more alternative polyadenylation sites. Choice of polyadenylation sites was suggested as one of the underlying mechanisms for generating longer/shorter transcript isoforms. Here, we demonstrate that mature mRNA transcripts can undergo additional cleavage and polyadenylation at a proximal internal site in the 3′-UTR, resulting in two stable, autonomous, RNA fragments: a coding sequence with a shorter 3′-UTR (body) and an uncapped 3′-UTR sequence downstream of the cleavage point (tail). Analyses of the human transcriptome has revealed thousands of such cleavage positions, suggesting a widespread post-transcriptional phenomenon producing thousands of stable 3′-UTR RNA tails that exist alongside their transcripts of origin. By analyzing the impact of microRNAs, we observed a significantly stronger effect for microRNA regulation at the body compared to the tail fragments. Our findings open a variety of future research prospects and call for a new perspective on 3′-UTR-dependent gene regulation. 1 The Lautenberg Center for Immunology and Cancer Research, IMRIC, Faculty of Medicine, The Hebrew University, Jerusalem 9112001, Israel. 2 Department of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, The Hebrew University, Jerusalem 9112001, Israel. 3 School of Computer Science and Engineering, The Hebrew University, Jerusalem 9190401, Israel. Correspondence and requests for materials should be addressed to Y.M. (email: [email protected]) or to T.K. (email: [email protected]) or to M.B. (email: [email protected]) NATURE COMMUNICATIONS | 8: 2029 | DOI: 10.1038/s41467-017-02099-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02099-7 he majority of mammalian genes undergo alternative observed transcripts are assembled from two independent RNA polyadenylation (APA) in the nucleus to generate RNA fragments, while in the nucleus these transcripts seem to originate T ′ isoforms with shorter 3 -UTRs. Accumulation of tran- from one long transcript. In addition, these results complement scripts with short 3′-UTRs is widespread across tissues and is the mRNA-seq results by demonstrating that the gap region positively correlated with cell proliferation1, membrane protein indeed represents a cleavage point. – localization2, and several diseases including cancer3 6. These findings support a model by which some transcripts 3′-UTRs serve as major docking platforms for regulatory could be cleaved at APA sites post-transcriptionally, resulting in proteins and RNAs, including microRNAs (miRNAs). Hence, it two autonomous RNA molecules. We therefore hypothesized that was initially assumed that APA increases the stability of the the downstream fragment should be uncapped at its 5′ end mRNAs and thus increases protein output7. However, recent (Fig. 1c). To test this hypothesis, RNA extracts from both the studies have demonstrated that the choice of APA sites has nucleus and cytoplasm were treated with terminator 5′-phos- minimal effects on stability and translational efficiency8, 9. Thus, phate-dependent exonuclease (TEX) that degrades uncapped the functional significance of this regulation has remained poorly RNA molecules. We then used qRT-PCR to quantify the levels of understood. corresponding upstream and downstream fragments. As Fig. 1d Recent results showed that mRNAs from defined neuron shows, TEX treatment of RNA results in a sharp decrease in populations exhibit widespread unbalanced expression of cognate cytoplasmic levels of the fragments downstream of the putative 3′-UTRs relative to respective upstream coding sequences (CDSs)10. cleavage point (compared to their “body” fragment) with limited The presence of a capped 3′-UTR transcript separately from its changes in the nucleus (p > 0.01). To further substantiate these associated CDS has also been documented by Mercer et al.11 for results we performed Northern blot analysis on Actb transcript (a some genes in humans, mice, and flies. Despite the potential high highly expressed gene with high mRNA-seq coverage and a impact of these results on the current understanding of the substantial gap at the 3′-UTR) using probes for either the gene transcriptome, no large-scale study of this phenomenon has been body or the tail (Supplementary Fig. 4a, b). Analysis using a body yet undertaken. probe indicates that most Actb copies are found in the short In this study, we show that thousands of mature poly(A) (cleaved) form and only a few copies in the canonical (uncleaved) mRNA transcripts could often be cleaved post-transcriptionally form. Both isoforms are TEX-insensitive. Conversely, the tail into two fragments: a “body” unit found upstream of the cleavage probes identified both the canonical and tail fragments in site and a downstream uncapped “tail” unit. By re-annotating the untreated RNA, with only the former present following TEX human transcriptome for novel cleavage positions, we show treatment. Overall, the results of the qRT-PCR and Northern blot stronger regulatory effects by miRNA sites in the body fragment experiments following TEX treatment indicate that mRNA can be compared to 3′-UTR sites downstream of the cleavage point. cleaved at 3′-UTR into (1) the gene body, containing the CDS with a shortened 3′-UTR and (2) one or more cleaved fragments, containing the downstream “tails” of the canonical transcript. Results These tail fragments are sensitive to TEX treatment, indicating 3′-UTR cleavage results in autonomous uncapped RNA frag- they are uncapped. Importantly, we observe higher sensitivity of ments. To systematically elucidate the process of 3′-UTR short- the tail fragments to TEX (compared to their body fragment) in ening, we turned to analyze published mRNA sequencing (RNA- the cytoplasm compared to the nucleus, indicating that these seq) data from naive mouse T cells, where 3′-UTR shortening is fragments are overrepresented in the cytoplasm in comparison to widespread and associated with the transition of the cells from the the nucleus. These results raise the possibility that cleavage occurs naive to activated state7. Manual examination of RNA-seq data12 post-transcriptionally, potentially by the canonical cleavage and revealed many genes with a distinctive gap in their read coverage polyadenylation machinery. along the 3′-UTR, adjacent to known APA sites13 (Fig. 1a and Finally, to further support the notion that tail fragments can be Supplementary Fig. 1). Moreover, these gaps were also frequently separated from the gene body, and to map the start site of the 3′- observed in published RNA-seq data from other tissues and UTR tail fragment at a single nucleotide resolution, we performed cells14, 15 (Fig. 1a and Supplementary Fig. 2a–o). These gaps are 5′-Rapid Amplification of cDNA Ends (5′-RACE) for tail characterized by unbalanced coverage of sequenced reads at their fragments of six selected genes that presented a substantial gap two sides, especially in brain tissue, suggesting that the gap may in APA site according to published RNA-seq data (Supplemen- represent a cleavage site that divides the transcript into two tary Figs. 4c and 5a–f). Sequencing of the 5′-RACE amplified independent fragments with different expression patterns. For products identified cleaved 3′-UTR fragments adjacently to APA example, Fig. 1a illustrates the RNA-seq read coverage in brain sites in all six samples. These results further support our model tissue for three genes: Ssr1 presents higher read coverage down- that 3′-UTR tails can be separated from their gene bodies, likely stream of the gap site; Bcl2 shows higher read coverage upstream through an APA mechanism. of the gap site; and Rab2a demonstrates a similar coverage on either side. To determine whether the observed gap represents an Analysis of 3′-UTRs identifies thousands of autonomous RNA authentic cleavage within the transcript, we used quantitative tails. In the following sections, we describe three large-scale real-time PCR (qRT-PCR) to compare the relative expression experiments to further support the suggested model at a levels of the regions upstream, downstream, and across the gap of transcriptome-wide scale. Ssr1, Bcl2, and Rab2a genes. Initially, total RNA extraction We developed three high-throughput methods to distinguish showed no substantial or minor change of expression between the between capped body fragments and uncapped tail fragments at a three amplicons (Supplementary Fig. 3). However, comparison of transcriptome-wide scale (Fig. 2a). On the one hand, to enrich for the relative expression of each region (upstream, downstream, capped body fragments, poly(A) selected RNA from U2OS cells and across the gap) between the cytoplasm and the nucleus, was either treated with TEX to degrade the uncapped tail showed a significant decrease of the amplicons corresponding to fragments or immunoprecipitated (IP) using anti-CAP antibody the gap region in the cytoplasm, while the upstream and (CAP IP). On the other hand, to enrich for tail fragments, poly(A) downstream amplicons demonstrated similar expression levels selected RNA was subjected to an in vitro capping system with (Fig. 1b). These