The key RNA sequence elements and protein factors necessary for 3' processing of polyadenylated mRNA precursors are well known. Recent studies, however, have significantly reshaped current models for ...the protein-RNA interactions involved in poly(A) site recognition, painting a picture more complex than previously envisioned and also providing new insights into regulation of this important step in gene expression. Here we review the recent advances in this area and provide a perspective for future studies.
Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3′-end for maturation. A crucial step in this process is the recognition of the AAUAAA ...polyadenylation signal (PAS), and the molecular mechanism of this recognition has been a long-standing problem. Here, we report the cryo-electron microscopy structure of a quaternary complex of human CPSF-160, WDR33, CPSF-30, and an AAUAAA RNA at 3.4-Å resolution. Strikingly, the AAUAAA PAS assumes an unusual conformation that allows this short motif to be bound directly by both CPSF-30 and WDR33. The A1 and A2 bases are recognized specifically by zinc finger 2 (ZF2) of CPSF-30 and the A4 and A5 bases by ZF3. Interestingly, the U3 and A6 bases form an intramolecular Hoogsteen base pair and directly contact WDR33. CPSF-160 functions as an essential scaffold and preorganizes CPSF-30 and WDR33 for high-affinity binding to AAUAAA. Our findings provide an elegant molecular explanation for how PAS sequences are recognized for mRNA 3′-end formation.
Alternative mRNA processing is a critical mechanism for proteome expansion and gene regulation in higher eukaryotes. The SR family proteins play important roles in splicing regulation. Intriguingly, ...mammalian genomes encode many poorly characterized SR-like proteins, including subunits of the mRNA 3′-processing factor CFIm, CFIm68 and CFIm59. Here we demonstrate that CFIm functions as an enhancer-dependent activator of mRNA 3′ processing. CFIm regulates global alternative polyadenylation (APA) by specifically binding and activating enhancer-containing poly(A) sites (PASs). Importantly, the CFIm activator functions are mediated by the arginine-serine repeat (RS) domains of CFIm68/59, which bind specifically to an RS-like region in the CPSF subunit Fip1, and this interaction is inhibited by CFIm68/59 hyper-phosphorylation. The remarkable functional similarities between CFIm and SR proteins suggest that interactions between RS-like domains in regulatory and core factors may provide a common activation mechanism for mRNA 3′ processing, splicing, and potentially other steps in RNA metabolism.
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•UGUA is a position-dependent enhancer for mRNA 3′ processing•CFIm is an enhancer-dependent activator of mRNA 3′ processing•The activator function of CFIm is mediated by its RS-like domains•mRNA 3′ processing and splicing share a common activation mechanism
Zhu et al. show that CFIm is an enhancer-dependent activator that promotes mRNA 3′-processing complex assembly. CFIm activator function requires the RS-like domains of CFIm68/59, and it involves a mechanism similar to SR protein-mediated splicing regulation, suggesting a unified activation mechanism for mRNA 3′ processing and splicing.
Cleavage stimulation factor 64 kDa (CstF64) is an essential pre-mRNA 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.
Recent studies have revealed widespread mRNA alternative polyadenylation (APA) in eukaryotes and its dynamic spatial and temporal regulation. APA not only generates proteomic and functional ...diversity, but also plays important roles in regulating gene expression. Global deregulation of APA has been demonstrated in a variety of human diseases. Recent exciting advances in the field have been made possible in a large part by high throughput analyses using newly developed experimental tools. Here I review the recent progress in global studies of APA and the insights that have emerged from these and other studies that use more conventional methods.
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 3' 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 3' 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 3' processing. Our data suggest that AAUAAA recognition in mammalian mRNA 3' processing is more complex than previously thought and involves multiple protein-RNA interactions.
In this paper, a feature termed as the postpeak value is proposed for the pulsed eddy current technique (PECT). Moreover, a method using the postpeak value is proposed to classify surface and reverse ...defects. A PECT system is built for verification purposes. Experiment results prove that the postpeak feature value has better performance than that of the traditional peak value in the case of reverse defect detection. In contrast, the peak value is better than the postpeak value in the case of surface defect detection. Experiment results also validate that the proposed classification algorithm has advantages: classification can be achieved in real time, the calculation process and results are easy to understand, and supervised training is unnecessary.
Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to ...characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%-50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3' untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation.
Cell fate transitions involve rapid gene expression changes and global chromatin remodeling, yet the underlying regulatory pathways remain incompletely understood. Here, we identified the ...RNA-processing factor Nudt21 as a novel regulator of cell fate change using transcription-factor-induced reprogramming as a screening assay. Suppression of Nudt21 enhanced the generation of induced pluripotent stem cells, facilitated transdifferentiation into trophoblast stem cells, and impaired differentiation of myeloid precursors and embryonic stem cells, suggesting a broader role for Nudt21 in cell fate change. We show that Nudt21 directs differential polyadenylation of over 1,500 transcripts in cells acquiring pluripotency, although only a fraction changed protein levels. Remarkably, these proteins were strongly enriched for chromatin regulators, and their suppression neutralized the effect of Nudt21 during reprogramming. Collectively, our data uncover Nudt21 as a novel post-transcriptional regulator of cell fate and establish a direct, previously unappreciated link between alternative polyadenylation and chromatin signaling.
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•shRNA screen identifies mRNA processing factor Nudt21 as a regulator of cell fate•Nudt21 knockdown enhances reprogramming but disrupts ESC/myeloid differentiation•Nudt21 suppression induces alternative polyadenylation for hundreds of transcripts•Chromatin regulators are enriched among Nudt21 targets important for reprogramming
Alternative polyadenylation exerts post-transcriptional control over cell fate decisions and pluripotency.
The majority of eukaryotic genes produce multiple mRNA isoforms by using alternative poly(A) sites in a process called alternative polyadenylation (APA). APA is a dynamic process that is highly ...regulated in development and in response to extrinsic or intrinsic stimuli. Mis-regulation of APA has been linked to a wide variety of diseases, including cancer, neurological and immunological disorders. Since the first example of APA was described 40 years ago, the regulatory mechanisms of APA have been actively investigated. Conventionally, research in this area has focused primarily on the roles of regulatory cis-elements and trans-acting RNA-binding proteins. Recent studies, however, have revealed important functions for epigenetic mechanisms, including DNA and histone modifications and higher-order chromatin structures, in APA regulation. Here we will discuss these recent findings and their implications for our understanding of the crosstalk between epigenetics and mRNA 3'-end processing.
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