The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5′ alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.
Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and—in certain situations—nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.
The SR proteins are essential factors that control the splicing of precursor mRNA by regulating multiple steps in spliceosome development. The prototypical SR protein ASF/SF2 contains two N-terminal RRMs (RRM1 and RRM2) and a 50-residue C-terminal RS (arginine-serine rich) domain that can be phosphorylated at numerous serines by the protein kinase SRPK1. The RS domain is further divided into N-terminal (RS1) and C-terminal (RS2) segments whose modification guides the nuclear localization of ASF/SF2. While previous studies revealed that SRPK1 phosphorylates RS1, regio- and temporal-specific control within the largely redundant RS domain is not well understood. To address this issue, engineered footprinting and single turnover experiments were performed to determine where and how SRPK1 initiates phosphorylation within the RS domain. The data show that local sequence elements in the RS domain control the strong kinetic preference for RS1 phosphorylation. SRPK1 initiates phosphorylation in a small region of serines (initiation box) in the middle of the RS domain at the C-terminal end of RS1 and then proceeds in an N-terminal direction. This initiation process requires both a viable docking groove in the large lobe of SRPK1 and one RRM (RRM2) on the N-terminal flank of the RS domain. Thus, while local RS/SR content steers regional preferences in the RS domain, distal contacts with SRPK1 guide initiation and directional phosphorylation within these regions.
protein kinase; regiospecificity; phosphorylation; splicing; SR protein
The posttranslational modification (PTM) in protein occurs in a regiospecific manner. In addition, the most commonly occurring PTMs involve similar residues in proteins such as acetylation, ubiquitylation, methylation and sumoylation at the lysine residue and phosphorylation and O-GlcNAc modification at serine/threonine residues. Thus, the possibility of modification sites where two such PTMs may occur in a mutually exclusive manner (ME-PTM) is much higher than known. A recent surge in the identification and the mapping of the commonly occurring PTMs in proteins has revealed that this is indeed the case. However, in what way such ME-PTM sites are regulated and what could be their relevance in the coordinated network of protein function remains to be known. To gain such potential insights in a biological context, we analyzed two most prevalent PTMs on the lysine residue by acetylation and ubiquitylation along with the most abundant PTM in proteins by phosphorylation among enzymes involved in glucose metabolism, a fundamental process in biology. The analysis of the PTM data sets has revealed two important clues that may be intrinsically associated with their regulation and function. First, the most commonly occurring PTMs by phosphorylation, acetylation and ubiquitylation are widespread and clustered in most of the enzymes involved in glucose metabolism; and the prevalence of phosphorylation sites correlates with the number of acetylation and ubiquitylation sites including the ME-modification sites. Second, the prevalence of ME-acetylation/ubiquitylation sites is exceptionally high among enzymes involved in glucose metabolism and have distinct pattern among the subset of enzymes of glucose metabolism such as glycolysis, tricarboxylic acid (TCA) cycle, glycogen synthesis, and the irreversible steps of gluconeogenesis. We hypothesize that phosphorylation including tyrosine phosphorylation plays an important role in the regulation of ME-acetylation/ubiquitylation sites and their similar pattern among the subset of functionally related proteins allows their coordinated regulation in the normal physiology. Similarly their coordinated dysregulation may underlie the disease processes such as reprogrammed metabolism in cancer, obesity, type 2 diabetes, and cardiovascular diseases. Our hypothesis provides an opportunity to understand the regulation of ME-PTMs in proteins and their relevance at the network level and is open for experimental validation.
posttranslational modification; phosphorylation; TCA cycle; glycolysis; glycogen synthesis; gluconeogenesis
SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, region-specific mapping of phosphorylation sites in ASF/SF2 was monitored as a function of the protein phosphatase PP1. We showed that ten phosphates added to the RS1 segment by SRPK1 are removed in a preferred N-to-C manner, directly opposing the C-to-N phosphorylation by SRPK1. Two N-terminal RNA recognition motifs (RRMs) in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RRMs protects against dephosphorylation suggesting that engagement of the SR protein with exonic splicing enhancers can regulate phosphoryl content in the RS domain. In addition to regulation by N-terminal domains, phosphorylation of the C-terminal portion of the RS domain (RS2) by the nuclear protein kinase Clk/Sty inhibits RS1 dephosphorylation and disrupts the directional mechanism. The data indicate that both RNA-protein interactions and phosphorylation in flanking sequences induce conformations of ASF/SF2 that increase the lifetime of phosphates in the RS domain.
protein kinase; protein phosphatase; phosphorylation; splicing; SR protein
Receptor interacting protein 140 (RIP140) undergoes extensive posttranslational modifications (PTMs), including phosphorylation, acetylation, arginine methylation, and pyridoxylation. PTMs affect its sub-cellular distribution, protein-protein interaction, and biological activity in adipocyte differentiation. Arginine methylation on Arg240, Arg650, and Arg948 suppresses the repressive activity of RIP140. Here we find that endogenous RIP140 in differentiated 3T3-L1 cells is also modified by lysine methylation. Three lysine residues, Lys591, Lys653, and Lys757 are mapped as potential methylation sites by mass spectrometry. Site-directed mutagenesis study shows that lysine methylation enhances its gene repressive activity. Mutation of lysine methylation sites enhances arginine methylation, while mutation on arginine methylation sites has little effect on its lysine methylation, suggesting a relationship between lysine methylation and arginine methylation. Kinetic analysis of PTMs of endogenous RIP140 in differentiated 3T3-L1 cells demonstrates sequential modifications on RIP140, initiated from constitutive lysine methylation, followed by increased arginine methylation later in differentiation. This study reveals a potential hierarchy of modifications, at least for lysine and arginine methylation, which bi-directionally regulate the functionality of a non-histone protein.
Mass spectrometry; Receptor interacting protein 140; Post-translational modification; Lysine methylation; Adipocytes differentiation
Chromatin disassembly and reassembly, mediated by histone chaperones such as anti-silencing function 1 (Asf1), are likely to accompany all nuclear processes that occur on the DNA template. In order to gain insight into the functional conservation of Asf1 across eukaryotes, we have replaced the budding yeast Asf1 protein with Drosophila Asf1 (dAsf1) or either of the two human Asf1 (hAsf1a and hAsf1b) counterparts. We found that hAsf1b is best able to rescue the growth defect of Saccharomyces cerevisiae lacking Asf1. Moreover, dAsf1 and hAsf1b but not hAsf1a can replace the role of yeast Asf1 in protecting against replicational stress and activating the PHO5 gene, while only hAsf1a can replace the role of Asf1 in protecting against double-stranded-DNA-damaging agents. Furthermore, it appears that the interaction between Asf1 and the DNA damage checkpoint protein Rad53 is not required for Asf1's role in maintaining genomic integrity. In addition to indicating the functional conservation of the Asf1 proteins across species, these studies suggest distinct roles for the two human Asf1 proteins.
Mammalian proteins expressed in Escherichia coli are used in a variety of applications. A major drawback in producing eukaryotic proteins in E.coli is that the bacteria lack most eukaryotic post-translational modification systems, including serine/threonine protein kinase(s). Here we show that a eukaryotic protein can be phosphorylated in E.coli by simultaneous expression of a mammalian protein kinase and its substrate. We show that in bacteria expressing SRPK1, ASF/SF2 becomes phosphorylated to a degree resembling native ASF/SF2 present in interphase HeLa cell nuclei. The E.coli phosphorylated ASF/SF2 is functional in splicing and, contrary to the unphosphorylated protein, soluble under native conditions.
Asf1 is a conserved histone H3/H4 chaperone that can assemble and disassemble nucleosomes and promote histone acetylation. Set2 is an H3 K36 methyltransferase. The functions of these proteins intersect in the context of transcription elongation by RNA polymerase II: both contribute to the establishment of repressive chromatin structures that inhibit spurious intragenic transcription. Here we characterize further interactions between budding yeast (Saccharomyces cerevisiae) Asf1 and Set2 using assays of intragenic transcription, H3/H4 posttranslational modification, coding region cross-linking of Asf1 and Set2, and cooccurrence of Asf1 and Set2 in protein complexes. We find that at some genes Asf1 and Set2 control chromatin metabolism as components of separate pathways. However, the existence of a low-abundance complex containing both proteins suggests that Asf1 and Set2 can more directly collaborate in chromatin regulation. Consistent with this possibility, we show that Asf1 stimulates Set2 occupancy of the coding region of a highly transcribed gene by a mechanism that depends on Asf1 binding to H3/H4. This function of Asf1 promotes the switch from di- to trimethylation of H3 K36 at that gene. These results support the view that Set2 function in chromatin metabolism can intimately involve histone chaperone Asf1.
Human DNA topoisomerase I, known for its DNA-relaxing activity, is possibly one of the kinases phosphorylating members of the SR protein family of splicing factors, in vivo. Little is known about the mechanism of action of this novel kinase. Using the prototypical SR protein SF2/ASF (SRp30a) as model substrate, we demonstrate that serine residues phosphorylated by topo I/kinase exclusively located within the most extended arginine-serine repeats of the SF2/ASF RS domain. Unlike other kinases such as cdc2 and SRPK1, which also phosphorylated serines at the RS domain, topo I/kinase required several SR dipeptide repeats. These repeats possibly contribute to a versatile structure in the RS domain thereby facilitating phosphorylation. Furthermore, far-western, fluorescence spectroscopy and kinase assays using the SF2/ASF mutants, demonstrated that kinase activity and binding were tightly coupled. Since the deletion of N-terminal 174 amino acids of Topo I destroys SF2/ASF binding and kinase activity but not ATP binding, we conclude that at least two distinct domains of Topo I are necessary for kinase activity: one in the C-terminal region contributing to the ATP binding site and the other one in the N-terminal region that allows binding of SF2/ASF.
Serine/arginine-rich splicing factor 1 (SRSF1), previously designated SF2/ASF, belongs to a family of SR proteins that regulate constitutive and alternative splicing. SRSF1 expression is increased in tumors from several tissues and elicits changes in key target genes involved in tumor genesis. Several protein kinases phosphorylate SRSF1, which regulates its localization and function. It is previously reported that protein kinase A (PKA) phosphorylates SRSF1, but the importance of this modification is not well characterized. Here, we show that PKA phosphorylates SRSF1 on serine 119 in vitro. Phosphorylation of SRSF1 on this site enhanced the RNA binding capacity of SRSF1 in vivo and reduced the protein’s capacity to activate splicing of the Minx transcript in vitro. We also confirm an interaction between SRSF1 and PKA Cα1 and demonstrate that this interaction is not dependent on serine 119 phosphorylation but requires active PKA Cα1. We conclude that PKA phosphorylation of SRSF1 at serine 119 regulates SFRS1-dependent RNA binding and processing but not its interaction with PKA.
pre-mRNA splicing regulation; SRSF1; PKA; phosphorylation
Acute lymphoblastic leukemia (ALL) is the most frequently-occurring malignant neoplasm in children, but the pathogenesis of the disease remains unclear. In a microarray assay using samples from 100 children with ALL, SFRS1 was found to be up-regulated. Serine/arginine-rich splicing factor 1 (SRSF1, also termed SF2/ASF), encoded by the SFRS1 gene, had been shown to be a pro-oncoprotein. Our previous study indicated that SRSF1 can be methylated by protein arginine methyltransferase 1 (PRMT1) in vitro; however, the biological function of SRSF1 and PRMT1 in pediatric ALL are presently unknown.
Matched, newly diagnosed (ND), complete remission (CR) and relapse (RE) bone marrow samples from 57 patients were collected in order to evaluate the expression patterns of SRSF1 and PRMT1. The potential oncogenic mechanism of SRSF1 and PRMT1 in leukemogenesis was also investigated.
We identified significant up-regulation of SRSF1 and PRMT1 in the ND samples. Importantly, the expression of SRSF1 and PRMT1 returned to normal levels after CR, but rebounded in the RE samples. Our observation that SRSF1 could predict disease relapse was of particular interest, although the expression patterns of SRSF1 and PRMT1 were independent of the cytogenetic subtypes. In pre-B-cell lines, both SRSF1 and PRMT1 expression could be efficiently attenuated by the clinical chemotherapy agents arabinoside cytosine (Ara-c) or vincristine (VCR). Moreover, SRSF1 and PRMT1 were associated with each other in leukemia cells in vivo. Knock-down of SRSF1 resulted in an increase in early apoptosis, which could be further induced by chemotherapeutics.
Our results indicate that SRSF1 serves as an anti-apoptotic factor and potentially contributes to leukemogenesis in pediatric ALL patients by cooperating with PRMT1.
Acute lymphoblastic leukemia; Splicing factor SRSF1; Protein arginine methyltransferase 1 (PRMT1); Alternative splicing; Arginine methylation
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.
RNA polymerase II, and specifically the C-terminal domain (CTD) of its largest subunit, has been demonstrated to play important roles in capping, splicing, and 3′ processing of mRNA precursors. But how the CTD functions in these reactions, especially splicing, is not well understood. To address some of the basic questions concerning CTD function in splicing, we constructed and purified two fusion proteins, a protein in which the CTD is positioned at the C terminus of the splicing factor ASF/SF2 (ASF-CTD) and an RS domain deletion mutant protein (ASFΔRS-CTD). Significantly, compared to ASF/SF2, ASF-CTD increased the reaction rate during the early stages of splicing, detected as a 20- to 60-min decrease in splicing lag time depending on the pre-mRNA substrate. The increased splicing rate correlated with enhanced production of prespliceosomal complex A and the early spliceosomal complex B but, interestingly, not the very early ATP-independent complex E. Additional assays indicate that the RS domain and CTD perform distinct functions, as exemplified by our identification of an activity that cooperates only with the CTD. Dephosphorylated ASFΔRS-CTD and a glutathione S-transferase-CTD fusion protein were both inactive, suggesting that an RNA-targeting domain and CTD phosphorylation were necessary. Our results provide new insights into the mechanism by which the CTD functions in splicing.
Histone H3 serine 10 phosphorylation is a hallmark of mitotic chromosomes but its full function remains to be elucidated. We report here that two SR protein splicing factors, SRp20 and ASF/SF2, associate with interphase chromatin, are released from hyperphosphorylated mitotic chromosomes but reassociate with chromatin late in M-phase. Inhibition of Aurora B kinase diminished histone H3 serine 10 phosphorylation and increased SRp20 and ASF/SF2 retention on mitotic chromosomes. Unexpectedly, we also found that HP1 proteins interact with ASF/SF2 in mitotic cells. Strikingly, siRNA-mediated knockdown of ASF/SF2 caused retention of HP1 proteins on mitotic chromatin. Finally, ASF/SF2-depleted cells released from a mitotic block displayed delayed G0/G1 entry, suggesting a functional consequence of these interactions. These findings underscore the evolving role of histone H3 phosphorylation and demonstrate a direct, functional and histone modification-regulated association of SRp20 and ASF/SF2 with chromatin.
The human immunodeficiency virus type 1 (HIV-1) potent transactivator Tat protein mediates pleiotropic effects on various cell functions. Posttranslational modification of Tat affects its activity during viral transcription. Tat binds to TAR and subsequently becomes acetylated on lysine residues by histone acetyltransferases. Novel protein-protein interaction domains on acetylated Tat are then established, which are necessary for both sustained transcriptional activation of the HIV-1 promoter and viral transcription elongation. In this study, we investigated the identity of proteins that preferentially bound acetylated Tat. Using a proteomic approach, we identified a number of proteins that preferentially bound AcTat, among which p32, a cofactor of splicing factor ASF/SF-2, was identified. We found that p32 was recruited to the HIV-1 genome, suggesting a mechanism by which acetylation of Tat may inhibit HIV-1 splicing needed for the production of full-length transcripts. Using Tat from different clades, harboring a different number of acetylation sites, as well as Tat mutated at lysine residues, we demonstrated that Tat acetylation affected splicing in vivo. Finally, using confocal microscopy, we found that p32 and Tat colocalize in vivo in HIV-1-infected cells.
Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs) and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK-mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases.
Posttranslational modifications (PTMs) are important strategies used by eukaryotic organisms to modulate their phenotypes. One of the well studied PTMs, arginine methylation, is catalyzed by protein arginine methyltransferases (PRMTs) with SAM as the methyl donor. The functions of PRMTs have been broadly studied in different biological processes and diseased states, but the molecular basis for arginine methylation is not well defined. In this study, we report the transient-state kinetic analysis of PRMT1 catalysis. The fast association and dissociation rates suggest that PRMT1 catalysis of histone H4 methylation follows a rapid equilibrium sequential kinetic mechanism. The data give direct evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, from the stopped-flow fluorescence measurements, we have identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. These results provide new insights into the mechanism of arginine methylation and the rational design of PRMT inhibitors.
PRMT1; arginine methylation; transient-state kinetics; conformational transition; fluorescent probe; stopped flow
The SR protein ASF/SF2, an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRM1-RRM2) and a C-terminal arginine-serine repeat region (RS domain). The serine protein kinase SRPK1 phosphorylates the RS domain at multiple serines using a directional (C-to-N-terminal) and processive mechanism, a process that directs the SR protein to the nucleus and influences protein-protein interactions associated with splicing function. To investigate how SRPK1 accomplishes this feat, the enzyme-substrate complex was analyzed using single and multi-turnover kinetic methods. Deletion studies revealed that while recognition of the RS domain by a docking groove on SRPK1 is sufficient to initiate the processive and directional mechanism, continued processive phosphorylation in the presence of building repulsive charge relies on the fine-tuning of contacts with the RRM1-RRM2 module. An electropositive pocket in SRPK1 that stabilizes newly phosphorylated serines enhanced processive phosphorylation of later serines. These data indicate that SRPK1 uses stable, yet highly flexible, protein-protein interactions to facilitate both early and late phases of processive phosphorylation of SR proteins.
kinase; kinetics; phosphorylation; splicing; SR protein
Human HIRA, ASF1a, ASF1b and CAF-1 are evolutionally conserved histone chaperones that form multiple functionally distinct chromatin assembly complexes, with roles linked to diverse nuclear process, such as DNA replication and formation of heterochromatin in senescent cells. We report the crystal structure of an ASF1a/HIRA heterodimer and a biochemical dissection of ASF1a's mutually exclusive interactions with HIRA and the p60 subunit of CAF-1. The HIRA B-domain forms an antiparallel β-hairpin that binds perpendicular to the strands of the β-sandwich of ASF1a, via β-sheet, salt-bridge and van der Waals contacts. The N- and C-terminal regions of ASF1a and ASF1b determine the different affinities of these two proteins for HIRA, by contacting regions outside the HIRA B-domain. CAF-1 p60 also employs B-domain-like motifs for binding to ASF1a, thereby competing with HIRA. Together, these studies begin to define the molecular determinants of assembly of functionally diverse macromolecular histone chaperone complexes.
Histone Deposition; Chromatin Regulation; Histone Chaperones; ASF1; HIRA; CAF-1
Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1–H3/H4 or H3/H4–DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)2 tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.
The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5′ half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.
Core histones are susceptible to a range of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation and ubiquitination, which play important roles in the epigenetic control of gene expression. Here, we observed an unusual discrepancy between MALDI-MS/MS and ESI-MS/MS on the methylation of trimethyllysine-containing peptides with residues 9–17 from human histone H3 and residues 73–83 from yeast histone H3. It turned out that the discrepancy could be attributed to an unusual methyl group migration from the side chain of trimethyllysine to the C-terminal arginine residue during peptide fragmentation, and this methyl group transfer only occurred for singly charged ions, but not for doubly charged ions. The methyl group transfer argument received its support from the results on the studies of the fragmentation of the ESI- or MALDI-produced singly charged ions of several synthetic trimethyllysine-bearing peptides. The results presented in this study highlighted that caution should be exerted while MS/MS of singly charged ions is employed to interrogate the PTMs of trimethyllysine-containing peptides.
Alternative splicing (AS) is the primary mechanism by which a limited number of protein coding genes can generate the proteome diversity. We have investigated the role of an alternative splicing factor (ASF), Sfrs1, an arginine/serine (SR) rich-protein family member, during retinal development. Here we report that loss of Sfrs1 function during embryonic retinal development had a profound effect such that it led to a small retina at birth. In addition, the retina underwent further degeneration in the postnatal period. Loss of Sfrs1 function resulted in the death of retinal neurons that were born during early and mid-embryonic development. Ganglion cells, cone photoreceptors, horizontal cells and amacrine cells were produced and initiated differentiation. However, these neurons subsequently underwent cell death through apoptosis. In contrast, Sfrs1 was not required for the survival of the neurons generated later, including later born amacrine cells, rod photoreceptors, bipolar cells and Müller glia. Our results highlight the requirement of Sfrs1-mediated AS for the survival of retinal neurons, with sensitivity defined by the window of time in which the neuron was generated. In all, this is the first description addressing the function of an ASF in vertebrate retinal development.
The covalent marking of proteins by methyl group addition to arginine residues can promote their recognition by binding partners or can modulate their biological activity. A small family of gene products that catalyze such methylation reactions in eukaryotes (PRMTs) work in conjunction with a changing cast of associated subunits to recognize distinct cellular substrates. These reactions display many of the attributes of reversible covalent modifications such as protein phosphorylation or protein lysine methylation; however, it is unclear to what extent protein arginine demethylation occurs. Physiological roles for protein arginine methylation have been established in signal transduction, mRNA splicing, transcriptional control, DNA repair, and protein translocation.