Cleavage stimulation factor 64 kDa (CstF64) is an essential premRNA 3′ processing factor and an important regulator of alternative polyadenylation (APA). Here we characterized CstF64-RNA interactions in vivo at the transcriptome level and investigated the role of CstF64 in global APA regulation through individual nucleotide resolution UV crosslinking and immunoprecipitation sequencing and direct RNA sequencing analyses. We observed highly specific CstF64-RNA interactions at poly(A) sites (PASs), and we provide evidence that such interactions are widely variable in affinity and may be differentially required for PAS recognition. Depletion of CstF64 by RNAi has a relatively small effect on the global APA profile, but codepletion of the CstF64 paralog CstF64τ leads to greater APA changes, most of which are characterized by the increased relative use of distal PASs. Finally, we found that CstF64 binds to thousands of dormant intronic PASs that are suppressed, at least in part, by U1 small nuclear ribonucleoproteins. Taken together, our findings provide insight into the mechanisms of PAS recognition and identify CstF64 as an important global regulator of APA.

AAUAAA is the most highly conserved motif in eukaryotic mRNA polyadenylation sites and, in mammals, is specifically recognized by the multisubunit CPSF (cleavage and polyadenylation specificity factor) complex. Despite its critical functions in mRNA 39 end formation, the molecular basis for CPSF-AAUAAA interaction remains poorly defined. The CPSF subunit CPSF160 has been implicated in AAUAAA recognition, but direct evidence has been lacking. Using in vitro and in vivo assays, we unexpectedly found that CPSF subunits CPSF30 and Wdr33 directly contact AAUAAA. Importantly, the CPSF30-RNA interaction is essential for mRNA 39 processing and is primarily mediated by its zinc fingers 2 and 3, which are specifically targeted by the influenza protein NS1A to suppress host mRNA 39 processing. Our data suggest that AAUAAA recognition in mammalian mRNA 39 processing is more complex than previously thought and involves multiple protein-RNA interactions.

mRNA alternative polyadenylation (APA) plays a critical role in post-transcriptional gene control and is highly regulated during development and disease. However, the regulatory mechanisms and functional consequences of APA remain poorly understood. Here, we show that an mRNA 3 0 processing factor, Fip1, is essential for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Fip1 promotes stem cell maintenance, in part, by activating the ESC-specific APA profiles to ensure the optimal expression of a specific set of genes, including critical self-renewal factors. Fip1 expression and the Fip1-dependent APA program change during ESC differentiation and are restored to an ESC-like state during somatic reprogramming. Mechanistically, we provide evidence that the specificity of Fip1-mediated APA regulation depends on multiple factors, including Fip1-RNA interactions and the distance between APA sites. Together, our data highlight the role for post-transcriptional control in stem cell self-renewal, provide mechanistic insight on APA regulation in development, and establish an important function for APA in cell fate specification.

BackgroundmicroRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs at the post-transcriptional stage. Several studies have shown that miRNAs modulate gene expression in mammalian cells by base pairing to complementary sites in the 3′-untranslated region (3′-UTR) of the target mRNAs.Methodology/Principal FindingsIn the present study, miR-24 was found to target fas associated factor 1(FAF1) by binding to its amino acid coding sequence (CDS) region, thereby regulating apoptosis in DU-145 cells. This result supports an augmented model whereby animal miRNAs can exercise their effects through binding to the CDS region of the target mRNA. Transfection of miR-24 antisense oligonucleotide (miR-24-ASO) also induced apoptosis in HGC-27, MGC-803 and HeLa cells.Conclusions/SignificanceWe found that miR-24 regulates apoptosis by targeting FAF1 in cancer cells. These findings suggest that miR-24 could be an effective drug target for treatment of hormone-insensitive prostate cancer or other types of cancers. Future work may further develop miR-24 for therapeutic applications in cancer biology.

SUMMARY Embryonic stem cell (ESC) self-renewal and differentiation are governed by a broad-ranging regulatory network. Although the transcriptional regulatory mechanisms involved have been investigated extensively, post-transcriptional regulation is still poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5, and is required for self-renewal at least in part by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, leading to decreased expression of pluripotency proteins that facilitates exit from self-renewal. THO is also important for the establishment of pluripotency, as its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicate that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and therefore uncover a role for this aspect of post-transcriptional regulation in stem cell fate specification.

AbstractmRNA 3′ end processing is an essential step in gene expression. It is well established that canonical eukaryotic pre-mRNA 3′ processing is carried out within a macromolecular machinery consisting of dozens of trans-acting proteins. However, it is unknown whether RNAs play any role in this process. Unexpectedly, we found that a subset of small nucleolar RNAs (snoRNAs) are associated with the mammalian mRNA 3′ processing complex. These snoRNAs primarily interact with Fip1, a component of cleavage and polyadenylation specificity factor (CPSF). We have functionally characterized one of these snoRNAs and our results demonstrated that the U/A-rich SNORD50A inhibits mRNA 3′ processing by blocking the Fip1-poly(A) site (PAS) interaction. Consistently, SNORD50A depletion altered the Fip1–RNA interaction landscape and changed the alternative polyadenylation (APA) profiles and/or transcript levels of a subset of genes. Taken together, our data revealed a novel function for snoRNAs and provided the first evidence that non-coding RNAs may play an important role in regulating mRNA 3′ processing.

mRNA 3′ processing is dynamically regulated spatially and temporally. However, the underlying mechanisms remain poorly understood. CstF64τ is a paralog of the general mRNA 3 ′ processing factor, CstF64, and has been implicated in mediating testis-specific mRNA alternative polyadenylation (APA). However, the functions of CstF64τ in mRNA 3 ′ processing have not been systematically investigated. We carried out a comprehensive characterization of CstF64τ and compared its properties to those of CstF64. In contrast to previous reports, we found that both CstF64 and CstF64τ are widely expressed in mammalian tissues, and their protein levels display tissue-specific variations. We further demonstrated that CstF64 and CstF64τ have highly similar RNA-binding specificities both in vitro and in vivo. CstF64 and CstF64τ modulate one another's expression and play overlapping as well as distinct roles in regulating global APA profiles. Interestingly, protein interactome analyses revealed key differences between CstF64 and CstF64τ, including their interactions with another mRNA 3 ′ processing factor, symplekin. Together, our study of CstF64 and CstF64τ revealed both functional overlap and specificity of these two important mRNA 3 ′ processing factors and provided new insights into the regulatory mechanisms of mRNA 3 ′ processing.

MicroRNAs (miRNAs), which are a newly identified class of small single-stranded non-coding RNAs, regulate their target genes via post-transcriptional pathway. It has been proved that miRNAs play important roles in many biological processes. To better understand miRNA function on type 2 diabetes, we used an oligonucleotide microarray to monitor miRNA expression profiles of Goto-Kakizaki (GK) and Wistar rats' skeletal muscle. It was found that seven miRNAs were downexpressed and two miRNAs were over-expressed in the muscle of GK rats. Among them, miR-24 showed the most prominent change. p38 MAPK, which is a direct target of miR-24, also showed expression difference. All the data give a clue that miR-24 might be associated with diabetes through down-regulation of p38 MAPK.