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Maria Carmo-Fonseca

Publications and source records attributed to Maria Carmo-Fonseca.

At least 19 recordsLinked to original sources
PubMed2006

Diversity of human U2AF splicing factors.

U2 snRNP auxiliary factor (U2AF) is an essential heterodimeric splicing factor composed of two subunits, U2AF(65) and U2AF(35). During the past few years, a number of proteins related to both U2AF(65) and U2AF(35) have been discovered. Here, we review the conserved structural features that characterize the U2AF protein families and their evolutionary emergence. We perform a comprehensive database search designed to identify U2AF protein isoforms produced by alternative splicing, and we discuss the potential implications of U2AF protein diversity for splicing regulation.

Alternative Splicing↗
PubMed2006

In vivo requirement of the small subunit of U2AF for recognition of a weak 3' splice site.

The U2 snRNP auxiliary factor (U2AF) is an essential splicing factor composed of two subunits, a large, 65-kDa subunit (U2AF(65)) and a small subunit, U2AF(35). U2AF(65) binds to the polypyrimidine tract upstream from the 3' splice site and promotes U2 snRNP binding to the pre-mRNA. Based on in vitro studies, it has been proposed that U2AF(35) plays a role in assisting U2AF(65) recruitment to nonconsensus polypyrimidine tracts. Here we have analyzed in vivo the roles of the two subunits of U2AF in the selection between alternative 3' splice sites associated with polypyrimidine tracts of different strengths. Our results reveal a feedback mechanism by which RNA interference (RNAi)-mediated depletion of U2AF(65) triggers the downregulation of U2AF(35). We further show that the knockdown of each U2AF subunit inhibits weak 3' splice site recognition, while overexpression of U2AF(65) alone is sufficient to activate the selection of this splice site. A variant of U2AF(65) lacking the interaction domain with U2AF(35) shows a reduced ability to promote this splicing event, suggesting that recognition of the weak 3' splice site involves the U2AF heterodimer. Furthermore, our data suggest that, rather than being required for splicing of all pre-mRNA substrates containing a weak polypyrimidine tract, U2AF(35) regulates the selection of weak 3' splice sites in a specific subset of cellular transcripts.

Base Sequence↗
PubMed2006

RNA interference knockdown of hU2AF35 impairs cell cycle progression and modulates alternative splicing of Cdc25 transcripts.

U2AF is a heterodimeric splicing factor composed of a large (U2AF65) and a small (U2AF35) subunit. In humans, alternative splicing generates two U2AF35 variants, U2AF35a and U2AF35b. Here, we used RNA interference to specifically ablate the expression of each isoform in HeLa cells. Our results show that knockdown of the major U2AF35a isoform reduced cell viability and impaired mitotic progression, leading to accumulation of cells in prometaphase. Microarray analysis revealed that knockdown of U2AF35a affected the expression level of approximately 500 mRNAs, from which >90% were underrepresented relative to the control. Among mRNAs underrepresented in U2AF35a-depleted cells we identified an essential cell cycle gene, Cdc27, for which there was an increase in the ratio between unspliced and spliced RNA and a significant reduction in protein level. Furthermore, we show that depletion of either U2AF35a or U2AF35b altered the ratios of alternatively spliced isoforms of Cdc25B and Cdc25C transcripts. Taken together our results demonstrate that U2AF35a is essential for HeLa cell division and suggest a novel role for both U2AF35 protein isoforms as regulators of alternative splicing of a specific subset of genes.

Alternative Splicing↗
PubMed2006

Abundance of the largest subunit of RNA polymerase II in the nucleus is regulated by nucleo-cytoplasmic shuttling.

Eukaryotic RNA polymerase II is a complex enzyme composed of 12 distinct subunits that is present in cells in low abundance. Transcription of mRNA by RNA polymerase II involves a phosphorylation/dephosphorylation cycle of the carboxyl-terminal domain (CTD) of the enzyme's largest subunit. We have generated stable murine cell lines expressing an alpha-amanitin-resistant form of the largest subunit of RNA polymerase II (RNA Pol II LS). These cells maintained transcriptional activity in the presence of alpha-amanitin, indicating that the exogenous protein was functional. We observed that over-expressed RNA Pol II LS was predominantly hypophosphorylated, soluble and accumulated in the cytoplasm in a CRM1-dependent manner. Our results further showed that the transcriptionally active form of RNA Pol II LS containing phosphoserine in position 2 of the CTD repeats was restricted to the nucleus and its levels remained remarkably constant. We propose that nucleo-cytoplasmic shuttling of RNA Pol II LS may provide a mechanism to control the pool of RNA polymerase subunits that is accessible for assembly of a functional enzyme in the nucleus.

Active Transport, Cell Nucleus↗
PubMed2006

Studies on the role of NonA in mRNA biogenesis.

The NonA protein of Drosophila melanogaster is an abundant nuclear protein that belongs to the DBHS (Drosophila behavior, human splicing) protein family. The DBHS proteins bind both DNA and RNA in vitro and have been involved in different aspects of gene expression, including pre-mRNA splicing, transcription regulation and nuclear retention of mRNA. We have used double-stranded RNA interference in Drosophila S2 cells to silence the expression of NonA and to investigate its role in mRNA biogenesis. We show that knockdown of NonA does not affect transcription nor splicing. We demonstrate that NonA forms a complex with the essential nuclear export factor NXF1 in an RNA-dependent manner. We have constructed stable S2 cell lines that express full-length and truncated NXF1 fused to GFP in order to perform fluorescence recovery after photobleaching experiments. We show that knockdown of NonA reduces the intranuclear mobility of NXF1-GFP associated with poly(A)(+) RNA in vivo, while the mobility of the truncated NXF1-GFP that does not bind RNA is not affected. Our data suggest that NonA facilitates the intranuclear mobility of mRNP particles.

Active Transport, Cell Nucleus↗
PubMed2006

Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors.

BACKGROUND: Pre-mRNA splicing is an essential step in gene expression that occurs co-transcriptionally in the cell nucleus, involving a large number of RNA binding protein splicing factors, in addition to core spliceosome components. Several of these proteins are required for the recognition of intronic sequence elements, transiently associating with the primary transcript during splicing. Some protein splicing factors, such as the U2 small nuclear RNP auxiliary factor (U2AF), are known to be exported to the cytoplasm, despite being implicated solely in nuclear functions. This observation raises the question of whether U2AF associates with mature mRNA-ribonucleoprotein particles in transit to the cytoplasm, participating in additional cellular functions. RESULTS: Here we report the identification of RNAs immunoprecipitated by a monoclonal antibody specific for the U2AF 65 kDa subunit (U2AF65) and demonstrate its association with spliced mRNAs. For comparison, we analyzed mRNAs associated with the polypyrimidine tract binding protein (PTB), a splicing factor that also binds to intronic pyrimidine-rich sequences but additionally participates in mRNA localization, stability, and translation. Our results show that 10% of cellular mRNAs expressed in HeLa cells associate differentially with U2AF65 and PTB. Among U2AF65-associated mRNAs there is a predominance of transcription factors and cell cycle regulators, whereas PTB-associated transcripts are enriched in mRNA species that encode proteins implicated in intracellular transport, vesicle trafficking, and apoptosis. CONCLUSION: Our results show that U2AF65 associates with specific subsets of spliced mRNAs, strongly suggesting that it is involved in novel cellular functions in addition to splicing.

Base Sequence↗
PubMed2005

Systematic genome-wide annotation of spliceosomal proteins reveals differential gene family expansion.

Although more than 200 human spliceosomal and splicing-associated proteins are known, the evolution of the splicing machinery has not been studied extensively. The recent near-complete sequencing and annotation of distant vertebrate and chordate genomes provides the opportunity for an exhaustive comparative analysis of splicing factors across eukaryotes. We describe here our semiautomated computational pipeline to identify and annotate splicing factors in representative species of eukaryotes. We focused on protein families whose role in splicing is confirmed by experimental evidence. We visually inspected 1894 proteins and manually curated 224 of them. Our analysis shows a general conservation of the core spliceosomal proteins across the eukaryotic lineage, contrasting with selective expansions of protein families known to play a role in the regulation of splicing, most notably of SR proteins in metazoans and of heterogeneous nuclear ribonucleoproteins (hnRNP) in vertebrates. We also observed vertebrate-specific expansion of the CLK and SRPK kinases (which phosphorylate SR proteins), and the CUG-BP/CELF family of splicing regulators. Furthermore, we report several intronless genes amongst splicing proteins in mammals, suggesting that retrotransposition contributed to the complexity of the mammalian splicing apparatus.

Animals↗
PubMed2005

SUMO-1 modification alters ADAR1 editing activity.

We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity.

Adenosine Deaminase↗
PubMed2005

In vivo aggregation properties of the nuclear poly(A)-binding protein PABPN1.

A broad range of degenerative diseases is associated with intracellular inclusions formed by toxic, aggregation-prone mutant proteins. Intranuclear inclusions constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD), a dominantly inherited disease caused by (GCG) repeat expansions in the gene that encodes for nuclear poly(A) binding protein (PABPN1). The mutation results in an extended polyalanine stretch that has been proposed to induce protein aggregation and formation of intranuclear inclusions. Here we show that normal PABPN1 is inherently aggregation-prone when exogenously expressed in either HeLa or myogenic C2 cells. Similar deposits of insoluble PABPN1 are formed by variant forms of the protein containing either a polyalanine expansion or a complete deletion of the polyalanine tract, indicating that the mutation responsible for OPMD is not essential for formation of PABPN1 inclusions. In contrast, interfering with any of the protein domains required for stimulation of poly(A) polymerase prevents the formation of inclusions. Most surprisingly, photobleaching experiments reveal that both normal and expanded PABPN1 molecules are not irreversibly sequestered into aggregates, but rather move rapidly in and out of the inclusions. These findings have important implications for the interpretation of OPMD model systems based on exogenous expression of PABPN1.

Animals↗
PubMed2004

Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes.

Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22 degrees C reduces the dextran diffusion rates by approximately 30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.

Dextrans↗
PubMed2004

Diversity of vertebrate splicing factor U2AF35: identification of alternatively spliced U2AF1 mRNAS.

U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF(35)) is encoded by a conserved gene designated U2AF1. Here we provide evidence for the existence of alternative vertebrate transcripts encoding different U2AF(35) isoforms. Three mRNA isoforms (termed U2AF(35)a-c) were produced by alternative splicing of the human U2AF1 gene. U2AF(35)c contains a premature stop codon that targets the resulting mRNA to nonsense-mediated mRNA decay. U2AF(35)b differs from the previously described U2AF(35)a isoform in 7 amino acids located at the atypical RNA Recognition Motif involved in dimerization with U2AF(65). Biochemical experiments indicate that isoform U2AF(35)b, which has been highly conserved from fish to man, maintains the ability to interact with U2AF(65), stimulates U2AF(65) binding to a pre-mRNA, and promotes U2AF splicing activity in vitro. Real time, quantitative PCR analysis indicates that U2AF(35)a is the most abundant isoform expressed in murine tissues, although the ratio between U2AF(35)a and U2AF(35)b varies from 10-fold in the brain to 20-fold in skeletal muscle. We propose that post-transcriptional regulation of U2AF1 gene expression may provide a mechanism by which the relative cellular concentration and availability of U2AF(35) protein isoforms are modulated, thus contributing to the finely tuned control of splicing events in different tissues.

Alternative Splicing↗
PubMed2004

Oculopharyngeal muscular dystrophy-like nuclear inclusions are present in normal magnocellular neurosecretory neurons of the hypothalamus.

Intranuclear inclusions composed of tubular filaments constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD). Autosomal dominant OPMD is caused by (GCG) repeat expansions in the gene that encodes for poly(A) binding protein nuclear 1 (PABPN1). The mutation results in the expansion of a polyalanine stretch in the N-terminus of the protein. It has been proposed that mutated PABPN1 induces protein aggregation, which in turn causes the formation of the filamentous nuclear inclusions. Here we report the presence of intranuclear inclusions composed of tubular filaments in oxytocin-producing neurons from normal rat hypothalamus. Like OPMD inclusions, the filamentous structures in neurosecretory neurons accumulate PABPN1, poly(A) RNA, ubiquitin and proteasomes. These inclusions do not contain members of Hsp40 and HDJ-2/DNAJ families of chaperones. The proportion of oxytocin-producing neurons that contain inclusions decreases during parturition and lactation (when synthesis and release of oxytocin is maximal) and increases at 1 day post-weaning (when occurs a drastic reduction in the production of the hormone). Thus, PABPN1 filaments in normal neurons are dynamic structures, the appearance of which correlate with changes in cellular activity. These data provide the first physiological evidence that polyalanine expansions are not essential to induce polymerization of PABPN1 into filamentous nuclear inclusions.

Animals↗
PubMed2004

A single RNA recognition motif in splicing factor ASF/SF2 directs it to nuclear sites of adenovirus transcription.

SR protein ASF/SF2 is a general pre-mRNA splicing factor as well as a regulator of alternative splicing. Data presented here show that ASF/SF2 is efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. At the intermediate stages of a productive infection, ASF/SF2 colocalizes with small nuclear ribonucleoprotein particles (snRNPs), splicing factors in ring-like structures surrounding viral replication centres and, at late stages of the infection, in enlarged speckles. Results presented here demonstrate that ASF/SF2 requires only one of the two RNA-recognition motifs (RRMs) present in the protein for its efficient recruitment to the ring-like structures, where viral pre-mRNAs are transcribed and processed, and that the arginine/serine-rich (RS) domain in ASF/SF2 is both redundant and insufficient for the translocation of the protein to active viral RNA polymerase II genes in adenovirus-infected cells.

Adenoviridae↗
PubMed2004

In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei.

Studies over the past years indicate that there is extensive coupling between nuclear export of mRNA and pre-mRNA processing. Here, we visualized the distribution of exon junction complex (EJC) proteins and RNA export factors relative to sites of abundant pre-mRNA synthesis in the nucleus. We analyzed both HeLa cells infected with adenovirus and murine erythroleukemia (MEL) cells stably transfected with the human beta-globin gene. Using in situ hybridization and confocal microscopy, we observe accumulation of EJC proteins (REF/Aly, Y14, SRm160, UAP56, RNPS1, and Magoh) and core spliceosome components (U snRNPs) at sites of transcription. This suggests that EJC proteins bind stably to pre-mRNA cotranscriptionally. No concentration of the export factors NXF1/TAP, p15, and Dbp5 was detected on nascent transcripts, arguing that in mammalian cells these proteins bind the mRNA shortly before or after release from the sites of transcription. These results also suggest that binding of EJC proteins to the mRNA is not sufficient to recruit TAP-p15, consistent with recent findings showing that the EJC does not play a crucial role in mRNA export. Contrasting to the results obtained in MEL cells expressing normal human beta-globin transcripts, mutant pre-mRNAs defective in splicing and 3'end processing do not colocalize with SRm160, REF, UAP56, or Sm proteins. This shows that the accumulation of EJC proteins at transcription sites requires efficient processing of the nascent pre-mRNAs, arguing that transcription per se is not sufficient for the stable assembly of the EJC.

Adenoviridae↗
PubMed2003

Regulation of adenovirus alternative RNA splicing correlates with a reorganization of splicing factors in the nucleus.

Subcellular compartmentalization of splicing factors has been proposed to play a role in the regulation of alternative splicing. In the present work we have addressed this issue using adenovirus-infected cells to visualize an alternative splicing switch at the single cell level. The adenovirus gene expression program requires the activation of distal alternative 3' splice sites during the late phase of infection in major late transcripts. We have established in situ hybridization conditions that allow for the specific detection of 3' alternatively spliced adenoviral mRNAs from the L1 transcript family. Results show that the switch from proximal to distal 3' splice sites correlates with a massive reorganization of splicing factors in the cell nucleus, involving their recruitment from nuclear speckles to sites of viral transcription. This observation raises the possibility that the subnuclear organization of splicing factors may be regulated in response to events that lead to the activation of alternative splicing programs.

Adenoviridae↗
PubMed2003

Dynamic association of RNA-editing enzymes with the nucleolus.

ADAR1 and ADAR2 are editing enzymes that deaminate adenosine to inosine in long double stranded RNA duplexes and specific pre-mRNA transcripts. Here, we show that full-length and N-terminally truncated forms of ADAR1 are simultaneously expressed in HeLa and COS7 cells owing to the usage of alternative starting methionines. Because the N-terminus of ADAR1 contains a nuclear export signal, the full-length protein localizes predominantly in the cytoplasm, whereas the N-terminally truncated forms are exclusively nuclear and accumulate in the nucleolus. ADAR2, which lacks a region homologous to the N-terminal domain of ADAR1, localizes exclusively to the nucleus and similarly accumulates in the nucleolus. Within the nucleolus, ADAR1 and ADAR2 co-localize in a novel compartment. Photobleaching experiments demonstrate that, in live cells, ADAR1 and ADAR2 are in constant flux in and out of the nucleolus. When cells express the editing-competent glutamate receptor GluR-B RNA, endogenous ADAR1 and ADAR2 de-localize from the nucleolus and accumulate at sites where the substrate transcripts accumulate. This suggests that ADAR1 and ADAR2 are constantly moving through the nucleolus and might be recruited onto specific editing substrates present elsewhere in the cell.

Adenosine Deaminase↗
PubMed2002

The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive.

After being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22 degrees C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes.

Active Transport, Cell Nucleus↗