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Biomedical subjects

James L Manley

Publications and source records attributed to James L Manley.

At least 37 records · Page 2Linked to original sources
PubMed2004

Dephosphorylated SRp38 acts as a splicing repressor in response to heat shock.

The cellular response to stresses such as heat shock involves changes in gene expression. It is well known that the splicing of messenger RNA precursors is generally repressed on heat shock, but the factors responsible have not been identified. SRp38 is an SR protein splicing factor that functions as a general repressor of splicing. It is activated by dephosphorylation and required for splicing repression in M-phase cells. Here we show that SRp38 is also dephosphorylated on heat shock and that this dephosphorylation correlates with splicing inhibition. Notably, depletion of SRp38 from heat-shocked cell extracts derepresses splicing, and adding back dephosphorylated SRp38 specifically restores inhibition. We further show that dephosphorylated SRp38 interacts with a U1 small nuclear ribonucleoprotein particle (snRNP) protein, and that this interaction interferes with 5'-splice-site recognition by the U1 snRNP. Finally, SRp38-deficient DT40 cells show an altered cell-cycle profile consistent with a mitotic defect; they are also temperature sensitive and defective in recovery after heat shock. SRp38 thus plays a crucial role in cell survival under stress conditions by inhibiting the splicing machinery.

Cell Cycle↗
PubMed2004

Cell signalling and the control of pre-mRNA splicing.

The transcripts of most metazoan protein-coding genes are alternatively spliced, but the mechanisms that are involved in the control of splicing are not well understood. Recent evidence supports the potential of both extra- and intracellular signalling to the splicing machinery as a means of regulating gene expression, and indicates that this form of gene control is widespread and mechanistically complex. However, important questions about these pathways need to be answered before this method of post-transcriptional regulation can be fully appreciated.

Animals↗
PubMed2004

Evidence that polyadenylation factor CPSF-73 is the mRNA 3' processing endonuclease.

Generation of the polyadenylated 3' end of an mRNA requires an endonucleolytic cleavage followed by synthesis of the poly(A) tail. Despite the seeming simplicity of the reaction, more than a dozen polypeptides are required, and nearly all appear to be necessary for the cleavage reaction. Because of this complexity, the identity of the endonuclease has remained a mystery. Here we present evidence that a component of the cleavage-polyadenylation specificity factor CPSF-73 is the long-sought endonuclease. We first show, using site-specific labeling and UV-cross-linking, that a protein with properties of CPSF-73 is one of only two polypeptides in HeLa nuclear extract to contact the cleavage site in an AAUAAA-dependent manner. The recent identification of CPSF-73 as a possible member of the metallo-beta-lactamase family of Zn(2+)-dependent hydrolytic enzymes suggests that this contact may identify CPSF-73 as the nuclease. Supporting the significance of the putative hydrolytic lactamase domain in CPSF-73, we show that mutation of key residues predicted to be required for activity in the yeast CPSF-73 homolog result in lethality. Furthermore, in contrast to long held belief, but consistent with properties of metallo-beta-lactamases, we show that 3' cleavage is metal-dependent, likely reflecting a requirement for tightly protein-bound Zn(2+). Taken together, the available data provide strong evidence that CPSF-73 is the 3' processing endonuclease.

Amino Acid Motifs↗
PubMed2003

Pin1 modulates the structure and function of human RNA polymerase II.

The C-terminal domain of the RNA polymerase (RNAP) II largest subunit (CTD) plays critical roles both in transcription of mRNA precursors and in the processing reactions needed to form mature mRNAs. The CTD undergoes dynamic changes in phosphorylation during the transcription cycle, and this plays a significant role in coordinating its multiple activities. But how these changes themselves are regulated is not well understood. Here we show that the peptidyl-prolyl isomerase Pin1 influences the phosphorylation status of the CTD in vitro by inhibiting the CTD phosphatase FCP1 and stimulating CTD phosphorylation by cdc2/cyclin B. This is reflected in vivo by accumulation of hypophosphorylated RNAP II in pin1-/- cells, and of a novel hyper-hyperphosphorylated form in cells induced to overexpress Pin1. This hyper-hyperphosphorylated form of RNAP II also accumulates in M-phase cells, in a Pin1-dependent manner, and associates specifically with Pin1. Functionally, we find that Pin1 overexpression specifically inhibits ongoing transcription of mRNA precursors in vivo and both transcription and RNAP II-stimulated pre-mRNA splicing in cell extracts. Pin1 thus plays a significant role in regulating RNAP II CTD structure and function.

Animals↗
PubMed2003

In vivo functional analysis of the histone 3-like TAF9 and a TAF9-related factor, TAF9L.

The majority of the TATA-binding protein (TBP)-associated factors (TAFs) that constitute transcription factor II D (TFIID) contain histone fold motifs (HFMs). Our previous results utilizing DT40 cells containing a conditional TAF9 allele indicated that the histone 3-like TAF9 is essential for cell viability but largely dispensable for general transcription. In this study, we investigated further the role of TAF9 structural domains in TFIID integrity and cell growth and the functions of a TAF9-related factor, TAF9L. We first show that TAF9 depletion severely disrupts TFIID, indicating that the observed ongoing transcription is initiated with at least partially TAF-free TATA-binding protein. We also provide evidence for specific roles of TAF HFMs, highlighting the functional significance of HFM specificity observed in vitro and, importantly, of the TAF9-histone 3 similarity. Although we provide evidence that TAF9 and TAF9L are partly redundant, RNA interference experiments suggest that TAF9L is essential for HeLa cell growth. Strikingly, we provide evidence that TAF9L plays a role in transcriptional repression and/or silencing.

Amino Acid Sequence↗
PubMed2003

Trypanosoma cruzi TcSRPK, the first protozoan member of the SRPK family, is biochemically and functionally conserved with metazoan SR protein-specific kinases.

A novel SR protein-specific kinase (SRPK) from the SRPK family was identified for the first time in a protozoan organism. The primary structure of the protein, named TcSRPK, presents a significant degree of identity with other metazoan members of the family. In vitro phosphorylation experiments showed that TcSRPK has the same substrate specificity relative to other SRPKs. TcSRPK was able to generate a mAb104-recognized phosphoepitope, a SRPK landmark. Expression of TcSRPK in different Schizosaccharomyces pombe strains lead to conserved phenotypes, indicating that TcSRPK is a functional homologue of metazoan SRPKs. In functional alternative splicing assays in vivo in HeLa cells, TcSRPK enhanced SR protein-dependent inclusion of the EDI exon of the fibronectin minigene. When tested in vitro, it inhibited splicing either on nuclear extracts or on splicing-deficient S100 extracts complemented with ASF/SF2. This inhibition was similar to that observed with human SRPK1. This work constitutes the first report of a member of this family of proteins and the existence of an SR-network in a protozoan organism. The implications in the origins and control of splicing are discussed.

Amino Acid Sequence↗
PubMed2003

Nucleotide binding by the Mdm2 RING domain facilitates Arf-independent Mdm2 nucleolar localization.

The RING domain of Mdm2 contains a conserved Walker A or P loop motif that is a characteristic of nucleotide binding proteins. We found that Mdm2 binds adenine-containing nucleotides preferentially and that nucleotide binding leads to a conformational change in the Mdm2 C terminus. Although nucleotide binding is not required for Mdm2 E3 ubiquitin ligase activity, we show that nucleotide binding-defective P loop mutants are impaired in p14(ARF)-independent nucleolar localization both in vivo and in vitro. Consistent with this, ATP-bound Mdm2 is preferentially localized to the nucleolus. Indeed, we identify a unique amino acid substitution in the P loop motif (K454A) that uncouples nucleolar localization and E3 ubiquitin ligase activity of Mdm2 and leads to upregulation of the E3 activity both in human cells and in Caenorhabditis elegans. We propose that nucleotide binding-facilitated nucleolar localization of Mdm2 is an evolutionarily conserved regulator of Mdm2 activity.

Active Transport, Cell Nucleus↗
PubMed2003

A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a relatively common neurodegenerative disease caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. Humans possess a linked, nearly identical gene, SMN2, which produces a functional SMN protein but at levels insufficient to compensate for loss of SMN1 (refs. 1,2). A C/T transition at position +6 in exon 7 is all that differentiates the two genes, but this is sufficient to prevent efficient exon 7 splicing in SMN2 (refs. 2,3). Here we show that the C/T transition functions not to disrupt an exonic splicing enhancer (ESE) in SMN1 (ref. 4), as previously suggested, but rather to create an exonic splicing silencer (ESS) in SMN2. We show that this ESS functions as a binding site for a known repressor protein, hnRNP A1, which binds to SMN2 but not SMN1 exon 7 RNA. We establish the physiological importance of these results by using small interfering RNAs to reduce hnRNP A protein levels in living cells and show that this results in efficient SMN2 exon 7 splicing. Our findings not only define a new mechanism underlying the inefficient splicing of SMN2 exon 7 but also illustrate more generally the remarkable sensitivity and precision that characterizes control of mRNA splicing.

Base Sequence↗
PubMed2003

Regulation and substrate specificity of the SR protein kinase Clk/Sty.

SR proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.

Blotting, Western↗
PubMed2003

Core promoter elements and TAFs contribute to the diversity of transcriptional activation in vertebrates.

Gene-specific transcriptional activation is a multistep process that requires numerous protein factors and DNA elements, including enhancers and the core promoter. To investigate the roles of core promoter elements in transcriptional activation in vertebrates, we examined expression and factor occupancy on representative promoters in chicken DT40 cells containing a conditional TATA binding protein (TBP)-associated factor 9 allele (TAF9). Characterized core elements, including TATA box-flanking regions and the downstream promoter element, were found to play significant roles in determining promoter strength, response to activators, and factor occupancy and recruitment. The requirement for TAF9 was found to be highly promoter specific, and TAF9 dependence and promoter occupancy were not always correlated. We also describe contrasting examples of factor recruitment and activation mechanisms at different promoters, highlighted by the nearly opposite mechanisms utilized by the simian virus 40 enhancer and p53. With the core promoters analyzed, the former functions by facilitating RNA polymerase II (RNAP II) recruitment to a preassembled TBP/TFIIB-containing scaffold and p53 strongly recruits TBP and TFIIB while RNAP II levels remain modest. Taken together, our results illustrate both the important roles of core promoter elements and the remarkable diversity that characterizes transcriptional activation mechanisms in vertebrates.

Animals↗
PubMed2003

ASAP, a novel protein complex involved in RNA processing and apoptosis.

Different isoforms of a protein complex termed the apoptosis- and splicing-associated protein (ASAP) were isolated from HeLa cell extract. ASAP complexes are composed of the polypeptides SAP18 and RNPS1 and different isoforms of the Acinus protein. While Acinus had previously been implicated in apoptosis and was recently identified as a component of the spliceosome, RNPS1 has been described as a general activator of RNA processing. Addition of ASAP isoforms to in vitro splicing reactions inhibits RNA processing mediated by ASF/SF2, by SC35, or by RNPS1. Additionally, microinjection of ASAP complexes into mammalian cells resulted in acceleration of cell death. Importantly, after induction of apoptosis the ASAP complex disassembles. Taken together, our results suggest an important role for the ASAP complexes in linking RNA processing and apoptosis.

Apoptosis↗
PubMed2003

Characterization of the catalytic activity of U2 and U6 snRNAs.

Removal of introns from pre-messenger RNAs in eukaryotes is carried out by the spliceosome, an assembly of a large number of proteins and five small nuclear RNAs (snRNAs). We showed previously that an in vitro transcribed and assembled base-paired complex of U2 and U6 snRNA segments catalyzes a reaction that resembles the first step of splicing. Upon incubation with a short RNA oligonucleotide containing the consensus sequence of the pre-mRNA branch site, the U2/U6 complex catalyzed a reaction between the 2' OH of a bulged adenosine and a phosphate in the catalytically important AGC triad of U6, leading to the formation of an X-shaped product, RNA X, apparently linked by an unusual phosphotriester bond. Here we characterize this splicing-related reaction further, showing that RNA X formation is an equilibrium reaction, and that the low yield of the reaction likely reflects an unfavorable equilibrium coefficient. Consistent with a phosphotriester linkage, RNA X is highly alkali-sensitive, but only mildly acid-sensitive. We also show that mutations in the AGC sequence of U6 can have significant effects on RNA X formation, further extending the similarities between splicing and RNA X formation. We also demonstrate that pseudouridylation of U2 enhances RNA X formation, and that U6 snRNA purified from nuclear extracts is capable of forming RNA X. Our data suggest that the ability to form RNA X might be an intrinsic property of spliceosomal snRNAs.

Base Sequence↗
PubMed2002

The SR protein SRp38 represses splicing in M phase cells.

SR proteins constitute a family of pre-mRNA splicing factors that play important roles in both constitutive and regulated splicing. Here, we describe one member of the family, which we call SRp38, with unexpected properties. Unlike other SR proteins, SRp38 cannot activate splicing and is essentially inactive in splicing assays. However, dephosphorylation converts SRp38 to a potent, general repressor that inhibits splicing at an early step. To investigate the cellular function of SRp38, we examined its possible role in cell cycle control. We show first that splicing, like other steps in gene expression, is inhibited in extracts of mitotic cells. Strikingly, SRp38 was found to be dephosphorylated specifically in mitotic cells, and we show that dephosphorylated SRp38 is required for the observed splicing repression.

Alternative Splicing↗
PubMed2002

Autoubiquitination of the BRCA1*BARD1 RING ubiquitin ligase.

The RING finger of BRCA1 confers ubiquitin ligase activity that is markedly enhanced when complexed with another RING-containing protein, BARD1, and is required for the function of this tumor suppressor protein in protecting genomic integrity. Here, we report that co-expression of BRCA1-(1-639) and BARD1 in bacteria can assemble a potent ubiquitin ligase activity. Purified BRCA1-(1-639)*BARD1 stimulated the Ubc5c-mediated monoubiquitination of histone H2A/H2AX in vitro, suggesting a possible role for BRCA1*BARD1 in modifying chromatin structure. Moreover, the truncated BRCA1*BARD1 complex exhibited efficient autoubiquitination activity in vitro capable of assembling non-lysine 48-linked polyubiquitin chains on both BRCA1-(1-639) and BARD1. When co-expressed in cells by transient transfection, the recombinant BRCA1-(1-300).BARD1 complex was found to be associated with polyubiquitin chains, suggesting that BRCA1-(1-300)*BARD1 was ubiquitinated in vivo as well. These results raise the possibility that BRCA1*BARD1 acts to assemble non-lysine 48-linked polyubiquitin chains that may serve as part of a signaling platform required for coordinating DNA repair-related events.

BRCA1 Protein↗
PubMed2002

Stability of a PKCI-1-related mRNA is controlled by the splicing factor ASF/SF2: a novel function for SR proteins.

Pre-mRNA splicing is a widely used regulatory mechanism for controlling gene expression, and a family of conserved proteins, SR proteins, participate in both constitutive and alternative splicing. Here we describe a novel function for the SR protein ASF/SF2. We used an embryonic chicken cDNA library to screen for differential mRNA expression in the chicken B-cell line DT40-ASF, expressing or not expressing ASF/SF2. Remarkably, out of 3 x 10(6) clones screened, only one, isolated several times independently, showed ASF/SF2-related differential expression. The isolated cDNA, referred to here as PKCI-r (for PKCI-related), is closely related to the protein kinase C interacting protein (PKCI-1) gene. Transcript levels were increased approximately sixfold in ASF/SF2-depleted cells compared with cells expressing ASF/SF2, indicating a negative role for the SR protein. Strikingly, inhibition of ASF/SF2 expression had no significant effect on PKCI-r splicing, or transcription, but markedly increased the half-life of PKCI-r mRNA (6.6-fold). Similarly, increased mRNA stability was also observed upon expression of exogenous PKCI-r mRNA in cells depleted of ASF/SF2. ASF/SF2 bound to a discrete region containing a purine-rich sequence in the 3' UTR of the PKCI-r transcript, and deletion of this region eliminated ASF/SF2-mediated regulation of transcript stability. Together these data indicate a novel, direct effect of ASF/SF2 on PKCI-r mRNA stability. Therefore, ASF/SF2, and perhaps other SR proteins, affects gene expression in vertebrate cells through regulation of mRNA stability as well as splicing.

3' Untranslated Regions↗