C. Sune - Academia.edu (original) (raw)

Papers by C. Sune

Research paper thumbnail of Protein-interaction modules that organize nuclear function: FF domains of CA150 bind the phosphoCTD of RNA polymerase II

Proceedings of the National Academy of Sciences, 2000

An approach for purifying nuclear proteins that bind directly to the hyperphosphorylated C-termin... more An approach for purifying nuclear proteins that bind directly to the hyperphosphorylated C-terminal repeat domain (CTD) of RNA polymerase II was developed and used to identify one human phosphoCTD-associating protein as CA150. CA150 is a nuclear factor implicated in transcription elongation. Because the hyperphosphorylated CTD is a feature of actively transcribing RNA polymerase II (Pol II), phosphoCTD (PCTD) binding places CA150 in a location appropriate for performing a role in transcription elongation-related events. Several recombinant segments of CA150 bound the PCTD. Predominant binding is mediated by the portion of CA150 containing six FF domains, compact modules of previously unknown function. In fact, small recombinant proteins containing the fifth FF domain bound the PCTD. PCTD binding is the first specific function assigned to an FF domain. As FF domains are found in a variety of nuclear proteins, it is likely that some of these proteins are also PCTD-associating proteins. Thus FF domains appear to be compact protein-interaction modules that, like WW domains, can be evolutionarily shuffled to organize nuclear function.

Research paper thumbnail of TCERG1 Regulates Alternative Splicing of the Bcl-x Gene by Modulating the Rate of RNA Polymerase II Transcription

Molecular and Cellular Biology, 2012

Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative sp... more Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNA polymerase II (RNAPII) may therefore have fundamental impacts in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-x s) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show that TCERG1 associates with the Bcl-x pre-mRNA. A transcription profile analysis revealed that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the proapoptotic Bcl-x S 5= splice site.

Research paper thumbnail of Human Transcription Elongation Factor CA150 Localizes to Splicing Factor-Rich Nuclear Speckles and Assembles Transcription and Splicing Components into Complexes through Its Amino and Carboxyl Regions

Molecular and Cellular Biology, 2006

The human transcription elongation factor CA150 contains three N-terminal WW domains and six cons... more The human transcription elongation factor CA150 contains three N-terminal WW domains and six consecutive FF domains. WW and FF domains, versatile modules that mediate protein-protein interactions, are found in nuclear proteins involved in transcription and splicing. CA150 interacts with the splicing factor SF1 and with the phosphorylated C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) through its WW and FF domains, respectively. WW and FF domains may, therefore, serve to link transcription and splicing components and play a role in coupling transcription and splicing in vivo. In the study presented here, we investigated the subcellular localization and association of CA150 with factors involved in pre-mRNA transcriptional elongation and splicing. Endogenous CA150 colocalized with nuclear speckles, and this was not affected either by inhibition of cellular transcription or by RNAPII CTD phosphorylation. FF domains are essential for the colocalization to speckles, while W...

Research paper thumbnail of The Transcription Elongation Factor CA150 Interacts with RNA Polymerase II and the Pre-mRNA Splicing Factor SF1

Molecular and Cellular Biology, 2001

CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcri... more CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcripts. The FF repeat domains of CA150 bind directly to the phosphorylated carboxyl-terminal domain of the largest subunit of RNAPII. We determined that this interaction is required for efficient CA150-mediated repression of transcription from the α4-integrin promoter. Additional functional determinants, namely, the WW1 and WW2 domains of CA150, were also required for efficient repression. A protein that interacted directly with CA150 WW1 and WW2 was identified as the splicing-transcription factor SF1. Previous studies have demonstrated a role for SF1 in transcription repression, and we found that binding of the CA150 WW1 and WW2 domains to SF1 correlated exactly with the functional contribution of these domains for repression. The binding specificity of the CA150 WW domains was found to be unique in comparison to known classes of WW domains. Furthermore, the CA150 binding site, within the ...

Research paper thumbnail of Induction of Transmissible Gastroenteritis Coronavirus-neutralizing Antibodies in vitro by Virus-specific T Helper Cell Hybridomas

Journal of General Virology, 1989

Three transmissible gastroenteritis (TGE) virus-specific T helper (Th) cell hybridomas have been ... more Three transmissible gastroenteritis (TGE) virus-specific T helper (Th) cell hybridomas have been generated from virus-primed BALB/c mice, by fusion with the thymoma BW5147. The hybridomas responded to purified u.v.-inactivated TGE virus with interleukin production and growth inhibition. TGE virus recognition by the hybridomas was restricted by the major histocompatibility complex : only splenocytes from syngeneic or semi-syngeneic mice were able to recognize the antigen. The three hybridomas were Thy 1.2 ÷, but did not express detectable levels of Lyt 1 or Lyt 2 antigens by fluorescent cell sorting analysis. Only one hybridoma (T. 1J. B5) expressed the L3T4 marker. These hybridomas had helper activity, as they were able to reconstitute in vitro the synthesis of TGE virus-specific antibodies by Th cell-depleted spleen cells from immune BALB/c mice. The antibodies that they induced specifically neutralized by 103-to 104-fold the infectivity of TGE virus, ruling out the possibility of inhibition of virus replication by interferon. These hybridomas could be very useful for identifying antigenic domains in TGE virus recognized by Th cells, which cooperate with B cells in the synthesis of neutralizing antibodies. antigenic sites (Correa et al., 1988). In contrast, very little information is available on the antigenic regions recognized by the T cell compartment. In this manuscript we describe the isolation of TGE virus-specific murine T helper (Th) cell hybridomas, which cooperated with B cells in the synthesis in vitro of virus-neutralizing antibodies. These hybridomas could be very useful in at least two areas: (i) the identification of T cell epitopes relevant in the induction of TGE virus-neutralizing antibodies and which could be included in a subunit vaccine containing B and T cell epitopes and (ii) the study of the antigenic variability of coronavirus with probes recognizing T cell epitopes.

Research paper thumbnail of Differential Effects of Sumoylation on Transcription and Alternative Splicing by Transcription Elongation Regulator 1 (TCERG1)

Journal of Biological Chemistry, 2010

Modification of proteins by small ubiquitin-like modifier (SUMO) is emerging as an important cont... more Modification of proteins by small ubiquitin-like modifier (SUMO) is emerging as an important control of transcription and RNA processing. The human factor TCERG1 (also known as CA150) participates in transcriptional elongation and alternative splicing of pre-mRNAs. Here, we report that SUMO family proteins modify TCERG1. Furthermore, TCERG1 binds to the E2 SUMO-conjugating enzyme Ubc9. Two lysines (Lys-503 and Lys-608) of TCERG1 are the major sumoylation sites. Sumoylation does not affect localization of TCERG1 to the splicing factor-rich nuclear speckles or the alternative splicing function of TCERG1. However, mutation of the SUMO acceptor lysine residues enhanced TCERG1 transcriptional activity, indicating that SUMO modification negatively regulates TCERG1 transcriptional activity. These results reveal a regulatory role for sumoylation in controlling the activity of a transcription factor that modulates RNA polymerase II elongation and mRNA alternative processing, which are discriminated differently by this post-translational modification. Splicing and transcriptional elongation are physically and functionally interconnected processes (1, 2). Although both processes can occur autonomously, their coupling and coordination may be important for regulation of gene expression. Coupling of these two processes may influence splicing and alternative splicing regulation. Indeed, promoter composition, transcriptional elongation efficiency, chromatin environment, and recruitment of specific coregulators to the transcriptional complex have been shown to affect alternative splicing decisions in a number of experimental systems (3-6).

Research paper thumbnail of Transcription Elongation Regulator 1 Is a Co-integrator of the Cell Fate Determination Factor Dachshund Homolog 1

Journal of Biological Chemistry, 2010

Research paper thumbnail of Promoter Influences Transcription Elongation: TATA-BOX ELEMENT MEDIATES THE ASSEMBLY OF PROCESSIVE TRANSCRIPTION COMPLEXES RESPONSIVE TO CYCLIN-DEPENDENT KINASE 9

Journal of Biological Chemistry, 2008

Research paper thumbnail of The FF4 and FF5 Domains of Transcription Elongation Regulator 1 (TCERG1) Target Proteins to the Periphery of Speckles

Journal of Biological Chemistry, 2012

Background: Coordinated transcription and splicing occurs at the periphery of speckles. Results: ... more Background: Coordinated transcription and splicing occurs at the periphery of speckles. Results: The FF4 and FF5 domains of transcription elongation regulator 1 (TCERG1) form a structural unit that directs proteins to the periphery of speckles. Conclusion: The FF4 and FF5 domains constitute a novel speckle periphery-targeting signal. Significance: This speckle periphery-targeting signal might participate in the coordination of transcription and splicing.

Research paper thumbnail of A transmissible gastroenteritis coronavirus nucleoprotein epitope elicits T helper cells that collaborate in the in vitro antibody synthesis to the three major structural …

Research paper thumbnail of Mechanisms of transmissible gastroenteritis coronavirus neutralization

Virology, 1990

Transmissible gastroenteritis virus (TGEV) was neutralized more than 10(9)-fold with antibodies o... more Transmissible gastroenteritis virus (TGEV) was neutralized more than 10(9)-fold with antibodies of a single specificity [monoclonal antibodies (MAbs)]. Most of the virus was neutralized in the first 2-3 min of a reversible reaction, which was followed by a second phase with a decreased neutralization rate and, in some cases, by a persistent fraction, which was a function of the MAb and of the antibody-to-virus ratio. Neutralization of TGEV is a specific event that requires the location of the epitope involved in the neutralization in the appropriate structural context, which is present in the wild-type virus but not in certain MAb escaping mutants. In neutralization of TGEV by binary combinations of MAbs specific for the same or for different antigenic sites, either no cooperation or a synergistic effect, respectively, was observed. Mechanisms of TGEV neutralization by MAbs were characterized at high, intermediate, and low antibody-to-virus ratios. Under these conditions, mainly three steps of the replication cycle were inhibited: binding of virus to the cell, internalization, and a step that takes place after internalization. In addition, virus aggregation could be responsible for the neutralization of 10 to 20% of virus infectivity.

Research paper thumbnail of Protein-interaction modules that organize nuclear function: FF domains of CA150 bind the phosphoCTD of RNA polymerase II

Proceedings of the National Academy of Sciences, 2000

An approach for purifying nuclear proteins that bind directly to the hyperphosphorylated C-termin... more An approach for purifying nuclear proteins that bind directly to the hyperphosphorylated C-terminal repeat domain (CTD) of RNA polymerase II was developed and used to identify one human phosphoCTD-associating protein as CA150. CA150 is a nuclear factor implicated in transcription elongation. Because the hyperphosphorylated CTD is a feature of actively transcribing RNA polymerase II (Pol II), phosphoCTD (PCTD) binding places CA150 in a location appropriate for performing a role in transcription elongation-related events. Several recombinant segments of CA150 bound the PCTD. Predominant binding is mediated by the portion of CA150 containing six FF domains, compact modules of previously unknown function. In fact, small recombinant proteins containing the fifth FF domain bound the PCTD. PCTD binding is the first specific function assigned to an FF domain. As FF domains are found in a variety of nuclear proteins, it is likely that some of these proteins are also PCTD-associating proteins. Thus FF domains appear to be compact protein-interaction modules that, like WW domains, can be evolutionarily shuffled to organize nuclear function.

Research paper thumbnail of TCERG1 Regulates Alternative Splicing of the Bcl-x Gene by Modulating the Rate of RNA Polymerase II Transcription

Molecular and Cellular Biology, 2012

Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative sp... more Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNA polymerase II (RNAPII) may therefore have fundamental impacts in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-x s) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show that TCERG1 associates with the Bcl-x pre-mRNA. A transcription profile analysis revealed that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the proapoptotic Bcl-x S 5= splice site.

Research paper thumbnail of Human Transcription Elongation Factor CA150 Localizes to Splicing Factor-Rich Nuclear Speckles and Assembles Transcription and Splicing Components into Complexes through Its Amino and Carboxyl Regions

Molecular and Cellular Biology, 2006

The human transcription elongation factor CA150 contains three N-terminal WW domains and six cons... more The human transcription elongation factor CA150 contains three N-terminal WW domains and six consecutive FF domains. WW and FF domains, versatile modules that mediate protein-protein interactions, are found in nuclear proteins involved in transcription and splicing. CA150 interacts with the splicing factor SF1 and with the phosphorylated C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) through its WW and FF domains, respectively. WW and FF domains may, therefore, serve to link transcription and splicing components and play a role in coupling transcription and splicing in vivo. In the study presented here, we investigated the subcellular localization and association of CA150 with factors involved in pre-mRNA transcriptional elongation and splicing. Endogenous CA150 colocalized with nuclear speckles, and this was not affected either by inhibition of cellular transcription or by RNAPII CTD phosphorylation. FF domains are essential for the colocalization to speckles, while W...

Research paper thumbnail of The Transcription Elongation Factor CA150 Interacts with RNA Polymerase II and the Pre-mRNA Splicing Factor SF1

Molecular and Cellular Biology, 2001

CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcri... more CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcripts. The FF repeat domains of CA150 bind directly to the phosphorylated carboxyl-terminal domain of the largest subunit of RNAPII. We determined that this interaction is required for efficient CA150-mediated repression of transcription from the α4-integrin promoter. Additional functional determinants, namely, the WW1 and WW2 domains of CA150, were also required for efficient repression. A protein that interacted directly with CA150 WW1 and WW2 was identified as the splicing-transcription factor SF1. Previous studies have demonstrated a role for SF1 in transcription repression, and we found that binding of the CA150 WW1 and WW2 domains to SF1 correlated exactly with the functional contribution of these domains for repression. The binding specificity of the CA150 WW domains was found to be unique in comparison to known classes of WW domains. Furthermore, the CA150 binding site, within the ...

Research paper thumbnail of Induction of Transmissible Gastroenteritis Coronavirus-neutralizing Antibodies in vitro by Virus-specific T Helper Cell Hybridomas

Journal of General Virology, 1989

Three transmissible gastroenteritis (TGE) virus-specific T helper (Th) cell hybridomas have been ... more Three transmissible gastroenteritis (TGE) virus-specific T helper (Th) cell hybridomas have been generated from virus-primed BALB/c mice, by fusion with the thymoma BW5147. The hybridomas responded to purified u.v.-inactivated TGE virus with interleukin production and growth inhibition. TGE virus recognition by the hybridomas was restricted by the major histocompatibility complex : only splenocytes from syngeneic or semi-syngeneic mice were able to recognize the antigen. The three hybridomas were Thy 1.2 ÷, but did not express detectable levels of Lyt 1 or Lyt 2 antigens by fluorescent cell sorting analysis. Only one hybridoma (T. 1J. B5) expressed the L3T4 marker. These hybridomas had helper activity, as they were able to reconstitute in vitro the synthesis of TGE virus-specific antibodies by Th cell-depleted spleen cells from immune BALB/c mice. The antibodies that they induced specifically neutralized by 103-to 104-fold the infectivity of TGE virus, ruling out the possibility of inhibition of virus replication by interferon. These hybridomas could be very useful for identifying antigenic domains in TGE virus recognized by Th cells, which cooperate with B cells in the synthesis of neutralizing antibodies. antigenic sites (Correa et al., 1988). In contrast, very little information is available on the antigenic regions recognized by the T cell compartment. In this manuscript we describe the isolation of TGE virus-specific murine T helper (Th) cell hybridomas, which cooperated with B cells in the synthesis in vitro of virus-neutralizing antibodies. These hybridomas could be very useful in at least two areas: (i) the identification of T cell epitopes relevant in the induction of TGE virus-neutralizing antibodies and which could be included in a subunit vaccine containing B and T cell epitopes and (ii) the study of the antigenic variability of coronavirus with probes recognizing T cell epitopes.

Research paper thumbnail of Differential Effects of Sumoylation on Transcription and Alternative Splicing by Transcription Elongation Regulator 1 (TCERG1)

Journal of Biological Chemistry, 2010

Modification of proteins by small ubiquitin-like modifier (SUMO) is emerging as an important cont... more Modification of proteins by small ubiquitin-like modifier (SUMO) is emerging as an important control of transcription and RNA processing. The human factor TCERG1 (also known as CA150) participates in transcriptional elongation and alternative splicing of pre-mRNAs. Here, we report that SUMO family proteins modify TCERG1. Furthermore, TCERG1 binds to the E2 SUMO-conjugating enzyme Ubc9. Two lysines (Lys-503 and Lys-608) of TCERG1 are the major sumoylation sites. Sumoylation does not affect localization of TCERG1 to the splicing factor-rich nuclear speckles or the alternative splicing function of TCERG1. However, mutation of the SUMO acceptor lysine residues enhanced TCERG1 transcriptional activity, indicating that SUMO modification negatively regulates TCERG1 transcriptional activity. These results reveal a regulatory role for sumoylation in controlling the activity of a transcription factor that modulates RNA polymerase II elongation and mRNA alternative processing, which are discriminated differently by this post-translational modification. Splicing and transcriptional elongation are physically and functionally interconnected processes (1, 2). Although both processes can occur autonomously, their coupling and coordination may be important for regulation of gene expression. Coupling of these two processes may influence splicing and alternative splicing regulation. Indeed, promoter composition, transcriptional elongation efficiency, chromatin environment, and recruitment of specific coregulators to the transcriptional complex have been shown to affect alternative splicing decisions in a number of experimental systems (3-6).

Research paper thumbnail of Transcription Elongation Regulator 1 Is a Co-integrator of the Cell Fate Determination Factor Dachshund Homolog 1

Journal of Biological Chemistry, 2010

Research paper thumbnail of Promoter Influences Transcription Elongation: TATA-BOX ELEMENT MEDIATES THE ASSEMBLY OF PROCESSIVE TRANSCRIPTION COMPLEXES RESPONSIVE TO CYCLIN-DEPENDENT KINASE 9

Journal of Biological Chemistry, 2008

Research paper thumbnail of The FF4 and FF5 Domains of Transcription Elongation Regulator 1 (TCERG1) Target Proteins to the Periphery of Speckles

Journal of Biological Chemistry, 2012

Background: Coordinated transcription and splicing occurs at the periphery of speckles. Results: ... more Background: Coordinated transcription and splicing occurs at the periphery of speckles. Results: The FF4 and FF5 domains of transcription elongation regulator 1 (TCERG1) form a structural unit that directs proteins to the periphery of speckles. Conclusion: The FF4 and FF5 domains constitute a novel speckle periphery-targeting signal. Significance: This speckle periphery-targeting signal might participate in the coordination of transcription and splicing.

Research paper thumbnail of A transmissible gastroenteritis coronavirus nucleoprotein epitope elicits T helper cells that collaborate in the in vitro antibody synthesis to the three major structural …

Research paper thumbnail of Mechanisms of transmissible gastroenteritis coronavirus neutralization

Virology, 1990

Transmissible gastroenteritis virus (TGEV) was neutralized more than 10(9)-fold with antibodies o... more Transmissible gastroenteritis virus (TGEV) was neutralized more than 10(9)-fold with antibodies of a single specificity [monoclonal antibodies (MAbs)]. Most of the virus was neutralized in the first 2-3 min of a reversible reaction, which was followed by a second phase with a decreased neutralization rate and, in some cases, by a persistent fraction, which was a function of the MAb and of the antibody-to-virus ratio. Neutralization of TGEV is a specific event that requires the location of the epitope involved in the neutralization in the appropriate structural context, which is present in the wild-type virus but not in certain MAb escaping mutants. In neutralization of TGEV by binary combinations of MAbs specific for the same or for different antigenic sites, either no cooperation or a synergistic effect, respectively, was observed. Mechanisms of TGEV neutralization by MAbs were characterized at high, intermediate, and low antibody-to-virus ratios. Under these conditions, mainly three steps of the replication cycle were inhibited: binding of virus to the cell, internalization, and a step that takes place after internalization. In addition, virus aggregation could be responsible for the neutralization of 10 to 20% of virus infectivity.