Partial loss of CFIm25 causes learning deficits and aberrant neuronal alternative polyadenylation.

We previously showed that NUDT21-spanning copy-number variations (CNVs) are associated with intellectual disability (Gennarino et al., 2015). However, the patients' CNVs also included other genes. To determine if reduced NUDT21 function alone can cause disease, we generated Nudt21+/- mice to mimic NUDT21-deletion patients. We found that although these mice have 50% reduced Nudt21 mRNA, they only have 30% less of its cognate protein, CFIm25. Despite this partial protein-level compensation, the Nudt21+/- mice have learning deficits, cortical hyperexcitability, and misregulated alternative polyadenylation (APA) in their hippocampi. Further, to determine the mediators driving neural dysfunction in humans, we partially inhibited NUDT21 in human stem cell-derived neurons to reduce CFIm25 by 30%. This induced APA and protein level misregulation in hundreds of genes, a number of which cause intellectual disability when mutated. Altogether, these results show that disruption of NUDT21-regulated APA events in the brain can cause intellectual disability.

Global Promotion of Alternative Internal Exon Usage by mRNA 3' End Formation Factors.

The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA. RBFOX2 and CPSF/SYMPK then function together to regulate binding of the early intron recognition factors U2AF and U1 small nuclear ribonucleoprotein particle (snRNP). Genome-wide analysis reveals that CPSF also mediates alternative splicing of many internal exons in the absence of RBFOX2. Accordingly, we show that CPSF/SYMPK is also a cofactor of NOVA2 and heterologous nuclear ribonucleoprotein A1 (HNRNPA1), RNA-binding proteins that also regulate alternative splicing. Collectively, our results reveal an unanticipated role for mRNA 3' end formation factors in global promotion of alternative splicing.

The Arabidopsis polyadenylation factor subunit CPSF30 as conceptual link between mRNA polyadenylation and cellular signaling.

Alternative polyadenylation plays important roles in growth processes in plants. Although the scope and significance of the phenomenon have been described to considerable extent, the mechanisms that govern differential poly(A) site selection remain active areas of investigation. Of particular interest are the means by which the factors that control differential poly(A) site choice are themselves activated and inhibited. In this review, the case is made that one particular Arabidopsis polyadenylation factor subunit, termed AtCPSF30, stands out as a conceptual link between cellular signaling pathways and differential poly(A) site choice.

Structure and function of pre-mRNA 5'-end capping quality control and 3'-end processing.

Messenger RNA precursors (pre-mRNAs) are produced as the nascent transcripts of RNA polymerase II (Pol II) in eukaryotes and must undergo extensive maturational processing, including 5'-end capping, splicing, and 3'-end cleavage and polyadenylation. This review will summarize the structural and functional information reported over the past few years on the large machinery required for the 3'-end processing of most pre-mRNAs, as well as the distinct machinery for the 3'-end processing of replication-dependent histone pre-mRNAs, which have provided great insights into the proteins and their subcomplexes in these machineries. Structural and biochemical studies have also led to the identification of a new class of enzymes (the DXO family enzymes) with activity toward intermediates of the 5'-end capping pathway. Functional studies demonstrate that these enzymes are part of a novel quality surveillance mechanism for pre-mRNA 5'-end capping. Incompletely capped pre-mRNAs are produced in yeast and human cells, in contrast to the general belief in the field that capping always proceeds to completion, and incomplete capping leads to defects in splicing and 3'-end cleavage in human cells. The DXO family enzymes are required for the detection and degradation of these defective RNAs.

Loss of function of the influenza A virus NS1 protein promotes apoptosis but this is not due to a failure to activate phosphatidylinositol 3-kinase (PI3K).

A panel of influenza A viruses encoding mutant NS1 proteins was created in which a number of NS1 functions, including interactions with dsRNA, PI3K, CPSF30 and PKR, were inhibited. Surprisingly, given previous reports that NS1 activates PI3K to prevent apoptosis, the mutant viruses rUd-Y89F and rUd-P164/7A that fail to activate PI3K did not induce any more apoptosis than wild-type virus in MRC-5 and A549 cells, even though these cells are highly sensitive to inducers of apoptosis. Induction of cell death by the apoptogenic rUd-184-8(P) virus could not be prevented by serum-mediated activation of PI3K/Akt. Neither infection of MRC-5 or A549 cells with wild-type virus nor constitutive expression of NS1 prevented cell death caused by apoptosis inducers, suggesting that NS1 is not directly anti-apoptotic. Our data suggest that the loss of a functionally intact NS1 protein promotes apoptosis, but this is not due to an inability to activate PI3K.

The tumor suppressor Cdc73 functionally associates with CPSF and CstF 3' mRNA processing factors.

The CDC73 tumor suppressor gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. Its product, the Cdc73 protein, is a component of the RNA polymerase II and chromatin-associated human Paf1 complex (Paf1C). Here, we show that Cdc73 physically associates with the cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF) complexes that are required for the maturation of mRNA 3' ends in the cell nucleus. Immunodepletion experiments indicate that the Cdc73-CPSF-CstF complex is necessary for 3' mRNA processing in vitro. Microarray analysis of CDC73 siRNA-treated cells revealed INTS6, a gene encoding a subunit of the Integrator complex, as an in vivo Cdc73 target. Cdc73 depletion by siRNA resulted in decreased INTS6 mRNA abundance, and decreased association of CPSF and CstF subunits with the INTS6 locus. Our results suggest that Cdc73 facilitates association of 3' mRNA processing factors with actively-transcribed chromatin and support the importance of links between tumor suppression and mRNA maturation.

Conserved motifs in both CPSF73 and CPSF100 are required to assemble the active endonuclease for histone mRNA 3'-end maturation.

In eukaryotes, the process of messenger RNA 3'-end formation involves endonucleolytic cleavage of the transcript followed by synthesis of the poly(A) tail. The complex machinery involved in this maturation process contains two proteins of the metallo-beta-lactamase (MBL) superfamily, the 73 and 100 kDa subunits of the cleavage and polyadenylation specificity factor (CPSF). By using an in vitro system to assess point mutations in these two mammalian proteins, we found that conserved residues from the MBL motifs of both polypeptides are required for assembly of the endonuclease activity that cleaves histone pre-mRNAs. This indicates that CPSF73 and CPSF100 act together in the process of maturation of eukaryotic pre-messenger RNAs, similar to other members of the MBL family, RNases Z and J, which function as homodimers.

Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

A hallmark of male germ cell gene expression is the generation by alternative polyadenylation of cell-specific mRNAs, many of which utilize noncanonical A(A/U)UAAA-independent polyadenylation signals. Cleavage factor I (CFIm), a component of the pre-mRNA cleavage and polyadenylation protein complex, can direct A(A/U)UAAA-independent polyadenylation site selection of somatic cell mRNAs. Here we report that the CFIm subunits NUDT21/CPSF5 and CPSF6 are highly enriched in mouse male germ cells relative to somatic cells. Both subunits are expressed from spermatogenic cell mRNAs that are shorter than the corresponding somatic transcripts. Complementary DNA sequencing and Northern blotting revealed that the shorter Nudt21 and Cpsf6 mRNAs are generated by alternative polyadenylation in male germ cells using proximal poly(A) signals. Both sets of transcripts contain CFIm binding sites within their 3'-untranslated regions, suggesting autoregulation of CFIm subunit formation in male germ cells. CFIm subunit mRNA and protein levels exhibit distinct developmental variation during spermatogenesis, indicating stage-dependent translational and/or posttranslational regulation. CFIm binding sites were identified near the 3' ends of numerous male germ cell transcripts utilizing A(A/U)UAAA-independent sites. Together these findings suggest that CFIm complexes participate in alternative polyadenylation directed by noncanonical poly(A) signals during spermatogenesis.

What nuclease cleaves pre-mRNA in the process of polyadenylation?

A transcript-specific cleavage by a large set of proteins is the first stage of eukaryotic pre-mRNA polyadenylation. The main participant of this reaction-endonuclease-has not been discovered until now. However, mammalian CPSF-30 and yeast Yth 1p proteins are known to be homologues to Drosophila Clipper (CLP) protein, which possesses endoribonucleolytic activity. In the N-terminal region, all three proteins contain five copies of the CCCH zinc finger motif associated with nucleolytic activity in the case of CLP. The literature data on these proteins are reviewed here. These data were shown not to contradict the hypothesis that CPSF-30 and its homologues are the actual nucleases that cleave pre-mRNA in the process of polyadenylation.