Serine/arginine-rich splicing factor 1 (SRSF1) also known as alternative splicing factor 1 (ASF1), pre-mRNA-splicing factor SF2 (SF2) or ASF1/SF2 is a protein that in humans is encoded by the SRSF1gene.[5] ASF/SF2 is an essential sequence specific splicing factor involved in pre-mRNA splicing.[6][7][8] SRSF1 is the gene that codes for ASF/SF2[9] and is found on chromosome 17. The resulting splicing factor is a protein of approximately 33 kDa.[10] ASF/SF2 is necessary for all splicing reactions to occur, and influences splice site selection in a concentration-dependent manner, resulting in alternative splicing.[7] In addition to being involved in the splicing process, ASF/SF2 also mediates post-splicing activities, such as mRNA nuclear export and translation.[11]
Structure
ASF/SF2 is an SR protein, and as such, contains two functional modules: an arginine-serine rich region (RS domain), where the bulk of ASF/SF2 regulation takes place, and two RNA recognition motifs (RRMs), through which ASF/SF2 interacts with RNA and other splicing factors.[12][13] These modules have different functions within general splicing factor function.[13]
NMR structure of the second RRM domain of ASF/SF2 based on the PDB: 2O3D coordinates.[14]
ASF/SF2 (left: red/orange/yellow) complexed with SRPK1 (right: blue/green/yellow) based on the 3BEG crystallographic coordinates.[12]
Splicing
ASF/SF2 is an integral part of numerous components of the splicing process. ASF/SF2 is required for 5’ splice site cleavage and selection, and is capable of discriminating between cryptic and authentic splice sites.[10] Subsequent lariat formation during the first chemical step of pre-mRNA splicing also requires ASF/SF2.[10] ASF/SF2 promotes recruitment of the U1 snRNP to the 5’ splice site, and bridges the 5’ and 3’ splice sites to facilitate splicing reactions.[8] ASF/SF2 also associates with the U2 snRNP.[15] During the reaction, ASF/SF2 promotes the use of intron proximal sites and hinders the use of intron distal sites, affecting alternative splicing.[16][17] Alternative splicing is affected by ASF/SF2 in a concentration-dependent manner; differing concentrations of ASF/SF2 is a mechanism for alternative splicing regulation, and will result in differing amounts of product isoforms.[6] ASF/SF2 accomplishes this regulation through direct or indirect binding to exonic splicing enhancer (ESE) sequences.[16]
Post-splicing
ASF/SF2, in the presence of elF4E, promotes the initiation of translation of ribosome-bound mRNA by suppressing the activity of 4E-BP and recruiting molecules for further regulation of translation.[11] ASF/SF2 interacts with the nuclear export protein TAP in a regulated manner, controlling the export of mature mRNA from the nucleus.[18] An increase in cellular ASF/SF2 also will increase the efficiency of nonsense-mediated mRNA decay (NMD), favoring NMD that occurs before mRNA release from the nucleus over NMD that occurs after mRNA export from the nucleus to the cytoplasm.[19] This shift in NMD caused by increased ASF/SF2 is accompanied by overall enhancement of the pioneer round of translation, through elF4E-bound mRNA translation and subsequent translationally active ribosomes, increased association of pioneer translation initiation complexes with ASF/SF2, and increased levels of active TAP.[19]
Regulation through phosphorylation
ASF/SF2 has the ability to be phosphorylated at the serines in its RS domain by the SR specific protein kinase, SRPK1.[13] SRPK1 and ASF/SF2 form an unusually stable complex of apparent Kd of 50nM.[12][18] SRPK1 selectively phosphorylates up to twelve serines in the RS domain of ASF/SF2 through a directional and processive mechanism, moving from the C terminus to the N terminus.[13] This multi-phosphorylation directs ASF/SF2 to the nucleus, influencing a number of protein-protein interactions associated with splicing.[13] ASF/SF2's function in export of mature mRNA from the nucleus is dependent on its phosphorylation state; dephosphorylation of ASF/SF2 facilitates binding to TAP,[13] while phosphorylation directs ASF/SF2 to nuclear speckles.[18] Both phosphorylation and dephosphorylation of ASF/SF2 are important and necessary for proper splicing to occur, as sequential phosphorylation and dephosphorylation marks the transitions between stages in the splicing process.[20] In addition, hypophosphorylation and hyperphosphorylation of ASF/SF2 by Clk/Sty can lead to inhibition of splicing.[13]
Biological importance
Stability and fidelity
ASF/SF2 is involved in genomic stability; it is thought that RNA Polymerase recruits ASF/SF2 to nascent RNA transcripts to impede formation of mutagenic DNA:RNA hybrid R-loop structures between the transcript and the template DNA.[8] In this way, ASF/SF2 is protecting cells from the potential deleterious effects of transcription itself.[8] ASF/SF2 is also implicated in cellular mechanisms to hinder exon skipping and to ensure splicing is occurring accurately and correctly.[10]
Development and growth
ASF/SF2 has been shown to have a critical function in heart development,[12] embryogenesis, tissue formation, cell motility, and cell viability in general.[21][22]
Clinical significance
SFRS1 is a proto-oncogene, and thus ASF/SF2 can act as an oncoprotein; it can alter the splicing patterns of crucial cell cycle regulatory genes and suppressor genes.[13] ASF/SF2 controls the splicing of various tumor suppressor genes, kinases, and kinase receptors, all of which have the potential to be alternatively spliced into oncogenic isoforms.[23] As such, ASF/SF2 is an important target for cancer therapy, as it is over-expressed in many tumors.[13]
Modifications and defects in the alternative splicing pathway are associated with a variety of human diseases.[24]
ASF/SF2 is involved in the replication of HIV-1, as HIV-1 needs a delicate balance of spliced and unspliced forms of its viral DNA.[25] ASF/SF2 action in the replication of HIV-1 is a potential target for HIV therapy.[25] ASF/SF2 is also implicated in the production of T cell receptors in Systemic Lupus Erythematosus, altering specific chain expression in T cell receptors through alternative splicing.[26][27]
^Masuyama K, Taniguchi I, Okawa K, Ohno M (Aug 2007). "Factors associated with a purine-rich exonic splicing enhancer sequence in Xenopus oocyte nucleus". Biochemical and Biophysical Research Communications. 359 (3): 580–5. doi:10.1016/j.bbrc.2007.05.144. PMID17548051.
^ abZhang X, Merkler KA, McLean MP (Jul 2008). "Characterization of regulatory intronic and exonic sequences involved in alternative splicing of scavenger receptor class B gene". Biochemical and Biophysical Research Communications. 372 (1): 173–8. doi:10.1016/j.bbrc.2008.05.007. PMID18477479.
^ abcMa CT, Velazquez-Dones A, Hagopian JC, Ghosh G, Fu XD, Adams JA (Feb 2008). "Ordered multi-site phosphorylation of the splicing factor ASF/SF2 by SRPK1". Journal of Molecular Biology. 376 (1): 55–68. doi:10.1016/j.jmb.2007.08.029. PMID18155240.
^Watanuki T, Funato H, Uchida S, Matsubara T, Kobayashi A, Wakabayashi Y, Otsuki K, Nishida A, Watanabe Y (Sep 2008). "Increased expression of splicing factor SRp20 mRNA in bipolar disorder patients". Journal of Affective Disorders. 110 (1–2): 62–9. doi:10.1016/j.jad.2008.01.003. PMID18281098.
^ abTange TØ, Kjems J (Sep 2001). "SF2/ASF binds to a splicing enhancer in the third HIV-1 tat exon and stimulates U2AF binding independently of the RS domain". Journal of Molecular Biology. 312 (4): 649–62. doi:10.1006/jmbi.2001.4971. PMID11575921.
^Moulton V, Perl M, Tsokos G (2008). "Alternative splicing factor/splicing factor 2 (ASF/SF2) regulates the expression of T cell receptor ζ chain". Clinical Immunology. 127: S95. doi:10.1016/j.clim.2008.03.266.
^ abcUmehara H, Nishii Y, Morishima M, Kakehi Y, Kioka N, Amachi T, Koizumi J, Hagiwara M, Ueda K (Feb 2003). "Effect of cisplatin treatment on speckled distribution of a serine/arginine-rich nuclear protein CROP/Luc7A". Biochemical and Biophysical Research Communications. 301 (2): 324–9. doi:10.1016/s0006-291x(02)03017-6. PMID12565863.