Symplekin and multiple other polyadenylation factors participate in 3'-end maturation of histone mRNAs.
(9/56)
Most metazoan messenger RNAs encoding histones are cleaved, but not polyadenylated at their 3' ends. Processing in mammalian cell extracts requires the U7 small nuclear ribonucleoprotein (U7 snRNP) and an unidentified heat-labile factor (HLF). We describe the identification of a heat-sensitive protein complex whose integrity is required for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor (CPSF), two subunits of the cleavage stimulation factor (CstF), and symplekin. Reconstitution experiments reveal that symplekin, previously shown to be necessary for cytoplasmic poly(A) tail elongation and translational activation of mRNAs during Xenopus oocyte maturation, is the essential heat-labile component. Thus, a common molecular machinery contributes to the nuclear maturation of mRNAs both lacking and possessing poly(A), as well as to cytoplasmic poly(A) tail elongation. (+info)
Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit.
(10/56)
The Arabidopsis (Arabidopsis thaliana) gene that encodes the probable ortholog of the 30-kD subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) is a complex one, encoding small (approximately 28 kD) and large (approximately 68 kD) polypeptides. The small polypeptide (AtCPSF30) corresponds to CPSF30 and is the focus of this study. Recombinant AtCPSF30 was purified from Escherichia coli and found to possess RNA-binding activity. Mutational analysis indicated that an evolutionarily conserved central core of AtCPSF30 is involved in RNA binding, but that RNA binding also requires a short sequence adjacent to the N terminus of the central core. AtCPSF30 was found to bind calmodulin, and calmodulin inhibited the RNA-binding activity of the protein in a calcium-dependent manner. Mutational analysis showed that a small part of the protein, again adjacent to the N terminus of the conserved core, is responsible for calmodulin binding; point mutations in this region abolished both binding to and inhibition of RNA binding by calmodulin. Interestingly, AtCPSF30 was capable of self-interactions. This property also mapped to the central conserved core of the protein. However, calmodulin had no discernible effect on the self-association. These results show that the central portion of AtCPSF30 is involved in a number of important functions, and they raise interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through calmodulin. (+info)
The CPSF30 binding site on the NS1A protein of influenza A virus is a potential antiviral target.
(11/56)
The emergence of influenza A viruses resistant to the two existing classes of antiviral drugs highlights the need for additional antiviral drugs, particularly considering the potential threat of a pandemic of H5N1 influenza A viruses. Here, we determine whether influenza A virus replication can be selectively inhibited by blocking the ability of its NS1A protein to inhibit the 3'-end processing of cellular pre-mRNAs, including beta interferon (IFN-beta) pre-mRNA. Pre-mRNA processing is inhibited via the binding of the NS1A protein to the cellular CPSF30 protein, and mutational inactivation of this NS1A binding site causes severe attenuation of the virus. We demonstrate that binding of CPSF30 is mediated by two of its zinc fingers, F2F3, and that the CPSF30/F2F3 binding site on the NS1A protein extends from amino acid 144 to amino acid 186. We generated MDCK cells that constitutively express epitope-tagged F2F3 in the nucleus, although at only approximately one-eighth the level of the NS1A protein produced during virus infection. Influenza A virus replication was inhibited in this cell line, whereas no inhibition was observed with influenza B virus, whose NS1B protein lacks a binding site for CPSF30. Influenza A virus, but not influenza B virus, induced increased production of IFN-beta mRNA in the F2F3-expressing cells. These results, which indicate that F2F3 inhibits influenza A virus replication by blocking the binding of endogenous CPSF30 to the NS1A protein, point to this NS1A binding site as a potential target for the development of antivirals directed against influenza A virus. (+info)
Direct interactions between subunits of CPSF and the U2 snRNP contribute to the coupling of pre-mRNA 3' end processing and splicing.
(12/56)
Eukaryotic pre-mRNAs are capped at their 5' ends, polyadenylated at their 3' ends, and spliced before being exported from the nucleus to the cytoplasm. Although the three processing reactions can be studied separately in vitro, they are coupled in vivo. We identified subunits of the U2 snRNP in highly purified CPSF and showed that the two complexes physically interact. We therefore tested whether this interaction contributes to the coupling of 3' end processing and splicing. We found that CPSF is necessary for efficient splicing activity in coupled assays and that mutations in the pre-mRNA binding site of the U2 snRNP resulted in impaired splicing and in much reduced cleavage efficiency. Moreover, we showed that efficient cleavage required the presence of the U2 snRNA in coupled assays. We therefore propose that the interaction between CPSF and the U2 snRNP contributes to the coupling of splicing and 3' end formation. (+info)
Defective RNA processing enhances RNA silencing and influences flowering of Arabidopsis.
(13/56)
Many eukaryotic cells use RNA-directed silencing mechanisms to protect against viruses and transposons and to suppress endogenous gene expression at the posttranscriptional level. RNA silencing also is implicated in epigenetic mechanisms affecting chromosome structure and transcriptional gene silencing. Here, we describe enhanced silencing phenotype (esp) mutants in Arabidopsis thaliana that reveal how proteins associated with RNA processing and 3' end formation can influence RNA silencing. These proteins were a putative DEAH RNA helicase homologue of the yeast PRP2 RNA splicing cofactor and homologues of mRNA 3' end formation proteins CstF64, symplekin/PTA1, and CPSF100. The last two proteins physically associated with the flowering time regulator FY in the 3' end formation complex AtCPSF. The phenotypes of the 3' end formation esp mutants include impaired termination of the transgene transcripts, early flowering, and enhanced silencing of the FCA-beta mRNA. Based on these findings, we propose that the ESP-containing 3' end formation complexes prevent transgene and endogenous mRNAs from entering RNA-silencing pathways. According to this proposal, in the absence of these ESP proteins, these RNAs have aberrant 3' termini. The aberrant RNAs would enter the RNA silencing pathways because they are converted into dsRNA by RNA-dependent RNA polymerases. (+info)
A serendipitous discovery that in situ proteolysis is essential for the crystallization of yeast CPSF-100 (Ydh1p).
(14/56)
The cleavage and polyadenylation specificity factor (CPSF) complex is required for the cleavage and polyadenylation of the 3'-end of messenger RNA precursors in eukaryotes. During structural studies of the 100 kDa subunit (CPSF-100, Ydh1p) of the yeast CPSF complex, it was serendipitously discovered that a solution that is infected by a fungus (subsequently identified as Penicillium) is crucial for the crystallization of this protein. Further analyses suggest that the protein has undergone partial proteolysis during crystallization, resulting in the deletion of an internal segment of about 200 highly charged and hydrophilic residues, very likely catalyzed by a protease secreted by the fungus. With the removal of this segment, yeast CPSF-100 (Ydh1p) has greatly reduced solubility and can be crystallized in the presence of a minute amount of precipitant. (+info)
Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.
(15/56)
Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including cleavage and polyadenylation at the 3'-end. Despite the characterization of many proteins that are required for the cleavage reaction, the identity of the endonuclease is not known. Recent analyses indicated that the 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF-73) might be the endonuclease for this and related reactions, although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 A resolution, complexed with zinc ions and a sulphate that might mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1) at 2.5 A resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-beta-lactamase domain and a novel beta-CASP (named for metallo-beta-lactamase, CPSF, Artemis, Snm1, Pso2) domain. The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3'-end-processing endonuclease. (+info)
Multiple anti-interferon actions of the influenza A virus NS1 protein.
(16/56)
The replication and pathogenicity of influenza A virus (FLUAV) are controlled in part by the alpha/beta interferon (IFN-alpha/beta) system. This virus-host interplay is dependent on the production of IFN-alpha/beta and on the capacity of the viral nonstructural protein NS1 to counteract the IFN system. Two different mechanisms have been described for NS1, namely, blocking the activation of IFN regulatory factor 3 (IRF3) and blocking posttranscriptional processing of cellular mRNAs. Here we directly compare the abilities of NS1 gene products from three different human FLUAV (H1N1) strains to counteract the antiviral host response. We found that A/PR/8/34 NS1 has a strong capacity to inhibit IRF3 and activation of the IFN-beta promoter but is unable to suppress expression of other cellular genes. In contrast, the NS1 proteins of A/Tx/36/91 and of A/BM/1/18, the virus that caused the Spanish influenza pandemic, caused suppression of additional cellular gene expression. Thus, these NS1 proteins prevented the establishment of an IFN-induced antiviral state, allowing virus replication even in the presence of IFN. Interestingly, the block in gene expression was dependent on a newly described NS1 domain that is important for interaction with the cleavage and polyadenylation specificity factor (CPSF) component of the cellular pre-mRNA processing machinery but is not functional in A/PR/8/34 NS1. We identified the Phe-103 and Met-106 residues in NS1 as being critical for CPSF binding, together with the previously described C-terminal binding domain. Our results demonstrate the capacity of FLUAV NS1 to suppress the antiviral host defense at multiple levels and the existence of strain-specific differences that may modulate virus pathogenicity. (+info)