Regulated polyadenylation of clam maternal mRNAs in vitro

During meiotic maturation of Spisula oocytes, maternal mRNAs undergo changes in translation and in the length of their poly(A) tails. In general, those mRNAs that are translationally activated, i.e., unmasked become polyadenylated, while deactivated mRNAs lose their poly(A) tails. The activated class of mRNAs encode ribonucleotide reductase, cyclins A and B and histone H3, while the proteins that stop being made include tubulin and actin. Previously, we demonstrated that mRNA-specific unmasking can be brought about in vitro by preventing the interaction of protein(s) with central portions of the 3′ noncoding regions (masking regions) of ribonucle-otide reductase and cyclin A mRNAs. In this report, we show that clam egg extracts are capable of sequence-specific polyadenylation of added RNAs since the 3′ untranslated regions (UTRs) of ribonu-cleotide reductase and histone H3 mRNAs are polyadenylated, while that of actin mRNA is not. In contrast, oocyte extracts, as in vivo, are essentially devoid of polyadenylation activity. We present an initial characterisation of the cis-acting sequences in the 3′ UTR of ribonucleotide reductase mRNA required for polyadenylation. The results suggest that the sequences for cytoplasmic polyadenylation are more complex and extensive than those determined in vertebrates and that they may partly overlap with the masking regions. © 1993 Wiley-Liss, Inc.     

Overlapping and distinct functions of CstF64 and CstF64τ in mammalian 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.     

A flexible linker region in Fip1 is needed for efficient mRNA polyadenylation

Synthesis of the poly(A) tail of mRNA in Saccharomyces cerevisiae requires recruitment of the polymerase Pap1 to the 3' end of cleaved pre-mRNA. This is made possible by the tethering of Pap1 to the Cleavage/Polyadenylation Factor (CPF) by Fip1. We have recently reported that Fip1 is an unstructured protein in solution, and proposed that it might maintain this conformation as part of CPF, when bound to Pap1. However, the role that this feature of Fip1 plays in 3' end processing has not been investigated. We show here that Fip1 has a flexible linker in the middle of the protein, and that removal or replacement of the linker affects the efficiency of polyadenylation. However, the point of tethering is not crucial, as a fusion protein of Pap1 and Fip1 is fully functional in cells lacking genes encoding the essential individual proteins, and directly tethering Pap1 to RNA increases the rate of poly(A) addition. We also find that the linker region of Fip1 provides a platform for critical interactions with other parts of the processing machinery. Our results indicate that the Fip1 linker, through its flexibility and protein/protein interactions, allows Pap1 to reach the 3' end of the cleaved RNA and efficiently initiate poly(A) addition.     

Importance of polyadenylation in the selective elimination of meiotic mRNAs in growing S. pombe cells

A number of meiosis‐specific mRNAs are initially weakly transcribed, but then selectively removed during fission yeast mitotic growth. These mRNAs harbour a region termed DSR (determinant of selective removal), which is recognized by the YTH family RNA‐binding protein Mmi1p. Mmi1p directs the destruction of these mRNAs in collaboration with nuclear exosomes. However, detailed molecular mechanisms underlying this process of selective mRNA elimination have remained elusive. In this study, we demonstrate the critical role of polyadenylation in this process. Two‐hybrid and genetic screens revealed potential interactions between Mmi1p and proteins involved in polyadenylation. Additional investigations showed that destruction of DSR‐containing mRNAs by exosomes required polyadenylation by a canonical poly(A) polymerase. The recruitment of Pab2p, a poly(A)‐binding protein, to the poly(A) tail was also necessary for mRNA destruction. In cells undergoing vegetative growth, Mmi1p localized with exosomes, Pab2p, and components of the polyadenylation complex in several patchy structures in the nucleoplasm. These patches may represent the sites for degradation of meiosis‐specific mRNAs with untimely expression.     

Fine-tuning of fgf8a expression through alternative polyadenylation has a selective impact on Fgf-associated developmental processes

The formation of distinct 3′UTRs through alternative polyadenylation is a mechanism of gene expression regulation that has been implicated in many physiological and pathological processes. However, its functions in the context of vertebrate embryonic development have been largely unaddressed, in particular with a gene-specific focus. Here we show that the most abundant 3′UTR for the zebrafish fgf8a gene in the developing embryo mediates a strong translational repression, when compared to a more sparsely used alternative 3′UTR, which supports a higher translation efficiency. By inducing a shift in the selection efficiency of the associated polyadenylation sites, we show a temporally and spatially specific impact of fgf8a 3′UTR usage on embryogenesis, in particular at late stages during sensory system development. In addition, we identified a previously undescribed role for Fgf signalling in the initial stages of superficial retinal vascularization. These results reveal a critical functional importance of gene-specific alternative 3′UTRs in vertebrate embryonic development.     

Distinct functions of maternal and somatic Pat1 protein paralogs

We previously identified Xenopus Pat1a (P100) as a member of the maternal CPEB RNP complex, whose components resemble those of P-(rocessing) bodies, and which is implicated in translational control in Xenopus oocytes. Database searches have identified Pat1a proteins in other vertebrates, as well as paralogous Pat1b proteins. Here we characterize Pat1 proteins, which have no readily discernable sequence features, in Xenopus oocytes, eggs, and early embryos and in human tissue culture cells. xPat1a and 1b have essentially mutually exclusive expression patterns in oogenesis and embryogenesis. xPat1a is degraded during meiotic maturation, via PEST-like regions, while xPat1b mRNA is translationally activated at GVBD by cytoplasmic polyadenylation. Pat1 proteins bind RNA in vitro, via a central domain, with a preference for G-rich sequences, including the NRAS 5′ UTR G-quadruplex-forming sequence. When tethered to reporter mRNA, both Pat proteins repress translation in oocytes. Indeed, both epitope-tagged proteins interact with the same components of the CPEB RNP complex, including CPEB, Xp54, eIF4E1b, Rap55B, and ePAB. However, examining endogenous protein interactions, we find that in oocytes only xPat1a is a bona fide component of the CPEB RNP, and that xPat1b resides in a separate large complex. In tissue culture cells, hPat1b localizes to P-bodies, while mPat1a-GFP is either found weakly in P-bodies or disperses P-bodies in a dominant-negative fashion. Altogether we conclude that Pat1a and Pat1b proteins have distinct functions, mediated in separate complexes. Pat1a is a translational repressor in oocytes in a CPEB-containing complex, and Pat1b is a component of P-bodies in somatic cells.