Srp20-Human Alternative Splicing Factor

From Proteopedia

Overview

Figure 1. SRp20 Domain Representation. Shown are the RRM (green), the TAP binding linker (red line) and SR-rich domain (blue).

Introduction

History

Splicing is one step in the process of RNA maturation that cuts out introns and joins exons together. Both the spliceosome, a complex of snRNAs (U1, U2, etc.), and splicing factors like SRp20 interact with intron consensus sequences in the pre-mRNA to regulate this process. Alternative splicing allows one mRNA molecule to produce numerous proteins that perform different functions in a cell by inclusion and exclusion of RNA sequences. There are two main families of splicing factors: Serine-Arginine rich (SR) proteins and heterogeneous nuclear RiboNucleoProteins (hnRNPs). The SRp20 protein belongs to the SR protein family. All SR proteins are defined by a RNA-binding domain at the N-terminus and a serine-arginine rich domain at the C-terminus^[2]. The discovery of this family started in the 1900s with the SF2 (SRp30a) protein and has since come to include twelve proteins, all of which act as splicing factors. SRp20 was first discovered in calf thymus when it was separated with several other SR proteins based on their molecular weight^[2]. The SRp20 protein, called X16 originally, was actually discovered first in an earlier paper studying different genes that change expression during B lymphocyte development^[1]. At the time, the protein was assumed to play a role in RNA processing and cellular proliferation, a finding that was later proved in further studies^[3][4]. The SRp20 protein has been shown to play a role in cancer progression and neurological disorders, specifically through alternative splicing. For example, SRp20 has been shown to play a role in alternative splicing of the Tau protein, an integral protein in the progression of Alzheimer’s disease^[1]. SRp20 has even been found to serve as a splicing factor for its own mRNA, influencing the inclusion of exon 4^[1]. Another function of SRp20 is its role in export of mRNA out of the nucleus, notably H2A histone mRNA export^[5].

Structure and Function

SRp20 bound to RNA ligand (PDB entry 2i2y) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image Structure Determination Attempts to determine the structure of native SRp20 had been largely unsuccessful due to the low solubility of the protein. This is likely due to the hydrophobic core of the RRM and exposed hydrophobic residues for RNA recognition on the β-sheets. As a solution, researchers removed the SR domain from the C terminus, leaving only the SRp20 RRM and a small arginine rich segment at the C terminus, then fused with a soluble of Streptococcal protein G to the N terminus of the protein, providing the first published structure of the SRp20 RRM via NMR. However, the solution of the structure via NMR, in addition to fusion with a globular tag, results in multiple possible conformations of the protein, meaning measurements such as bond angles, lengths, and substrate interactions are variable. Further, information concerning structural aspects of the SR domain are still limited to experimental data of protein function with certain mutations or deletions, and by comparison to sister proteins such as 9G8. To date, structure of the SR domain or the protein without the globular tag have not been solved, nor has a crystal structure for any part of the protein been determined[5]. Splicing Activity The splicing mechanism for SRp20 follows the normal eukaryotic mechanism, in which five different small nuclear ribonucleoproteins (snRNPs) bring the splice sites together in order to start the reaction. The free 3’OH nucleophile of adenosine in the intron attacks the phosphorus of phosphate creating a ring structure intron known as a lariat. [6]. Figure 2. Splicing mechanism for eukaryotes. The products include the final modified RNA sequence and the lariat which will be recycled. Figure 3. SRp20 works with U2 snRNP: Five snRNPs are needed in the eukaryotic splicing mechanism to facilitate the reaction. The free 2’OH in the 5’ splice site acts as the nucleophile to attack the phosphate of the first nucleotide in the 3’ splice site to release the lariat intron (Figure 2). Specifically, SRp20 and other SR proteins interact with the RNA ligand at the exonic splicing enhancer sequence at the beginning of the 3’ splice site adjacent to the intron being removed. SRp20 facilitates the interaction of the U2 snRNP with the RNA to continue the mechanism (Figure 3) The U2 snRNP must attach to the 3’ splice site to bring together the 5’ and 3’ splice sites (green). SRp20 (blue) facilitates binding of U2 to the 3’ splice site by binding to the exonic splicing enhancer sequence in the RNA at the backbone. U2 must bind before the other snRNPs can bind to continue the mechanism (Figure 3). Structural Highlights RNA Recognition Motif The SRp20 RRM (aa 1-86) contains a βαββαβ , common of many other RRMs. To explore substrate binding, researchers used a 4 base RNA ligand with sequence CAUC, which matches the SRp20 recognition sequence found in the corresponding H2A histone mRNA. The RNA bases each onto an aromatic side chain protruding from one of the SRp20 β-sheets, forming the primary interactions that allow the substrate to bind to the protein. In particular, C1 on Y13 in β1, stacks on F50 in β3, and F48 of β3 sits in between the sugar rings of C1 and A2. It should also be noted that A2 adopts an irregular conformation when bound to the RRM, something that was previously observed only for guanine in the 2 position. U3 onto F48 in β3, as W40 and A42 in β2. However, when bound, U3 out of line in comparison to the rest of the substrate. C4 partially stacks over , and also forms hydrogen between the C4 amino group, A2 2’ oxygen, and a main chain phosphate oxygen. While all 4 bases form a number of hydrophobic stacking interactions, alteration to the last 3 bases of the substrate sequence does not significantly impact binding affinity, while the C to G mutation of C1 results in a 10-fold decrease in binding affinity. This suggests that C1 interacts specifically with the protein, while positions 2-4 interact nonspecifically. The SRp20 RRM is able to recognize C1 with high specificity primarily through 4 : from the C1 amino protons to Leu80 backbone carbonyl oxygen and to Glu79 side-chain carboxyl oxygen, from C1 N3 to Asn82 amide, and from C1 O2 to Ser81 side chain hydroxyl group. The semi-specific RNA recognition is a mechanism which reduces evolutionary pressure on bound mRNA by increasing the number of possible RNA recognition sequences. As a result, tolerance for possible mutation in the RNA sequence is increased, meaning SRp20 can bind a diverse range of substrates, or even to original substrates that were mutated during replication (eg. H2A mRNA with a point mutation). Therefore, this mechanism increases organism survival chance by reducing the probability of physiological impact as a result of certain mutations[5]. Tip Associated Protein Binding Domain In addition to RNA recognition and alternative splicing functions, SRp20 has been shown to associate with Tip Associated Protein (TAP), an mRNA export factor, to promote transport of bound mRNA out of the nucleus for eventual translation. In particular, SRp20 promotes the export of H2A histone mRNA by binding the CAUC consensus sequence on the mRNA and TAP. Previous experiments show that SRp20 binding TAP is dependent on the presence of both the and a short arginine-rich C-terminal segment after the RRM (aa 1-83 and 84-90 respectively) (Figure 1). Previous research also shows that mutation of any one of the three arginine residues between residues 84-90 to glutamate prevents TAP binding, indicative of the importance of these arginine residues in TAP association. The structure is only solved to residue 86 so only the is present. The same study also found that transfer of the TAP-binding motif to a non-functional REF2 RRM still allowed for TAP binding and nuclear export of the target protein, suggesting that not only is the TAP binding motif transferable, but it also does not depend on interaction with the host RRM to retain function[5]. SR Domain While the RNA-protein interaction occurs at the RRMs, SR domains are typically responsible for the recruitment of other proteins that act in the splicing mechanism. The serines within the SR domain are phosphorylated by kinases within the cell to direct them to pre-mRNA sites. Phosphorylation acts as a recruiting tool for the SRp20 protein and other SR proteins to promote splicing. However, some SR proteins have shown that phosphorylation actually leads to a decrease in splicing, as in the SRp38 protein undergoing heat shock. Research has recently revealed that serines are dephosphorylated as splicing continues, indicating how much of the mRNA strand is being spliced. This suggests that dephosphorylation serves as a signal to the cell that the mRNA is ready to be exported out of the nucleus. Rephosphorylation then appears to trigger the SR protein to enter back into the nucleus. The SR domain also appears to modulate mRNA stability, though different SR proteins have been shown to have different effects. Some SR proteins appear to increase the sensitivity of certain mRNA sequences to degradation; this degradation appears to be controlled by SR domain interactions with the 3’ UTR of the pre-mRNA. These effects are not present without the SR domain of the protein. The destabilizing effect of SRp20 specifically has yet to be studied[5]. Comparison of the 9G8 and SRp20 RRM Domains 9G8 is another SR protein that is 80% similar in amino acid sequence to SRp20. They are two of the smallest proteins in the SR family and both contain an RRM that promotes export of mRNA through interaction with the TAP protein. The only other protein shown to promote transport through the TAP protein is SF2, one of the first SR proteins discovered[7]. Both RRMs adopt the βαββαβ pattern and interact with RNA with limited selectivity, and therefore recognize many different RNA sequences. The 9G8 RRM contains a large hydrophobic core on its B-sheet. Observing the 9G8 protein has proved useful in understanding SRp20's less stable protein structure. In fact, one of the only significant structural differences between the two proteins lies in the loops between a-helix 2 and B-4 where several amino acids are not conserved. Aside from the RRM, both proteins have one SR-rich domain although 9G8 includes about 40 more amino acids this domain[1]. Figure 5. SRp20 and 9G8 proteins. The PDB file for SRp20 (left; PDB file: 2i2y) does not include the SR-rich domain but 9G8 (right; PDB file: 2hvz) does. Boxed image shows an RSR region in the 9G8 protein with oxygens highlighted in red. Images taken from PyMol software.jpg Within the bigger 9G8 protein, there is a zinc knuckle that allows for binding of pyrimidine-rich RNA sequences. This zinc knuckle is not present in SRp20, allowing the protein to bind to more purine-rich sequences[7]. Not only are these two proteins similar, but they also play similar roles in mRNA export out of the nucleus. Both move continuously between the nucleus and cytoplasm which requires phosphorylation of its serine residues located in the SR-rich domain. Serine phosphorylation has been shown to have great importance in the proper functioning of many SR proteins[6].

Medical Significance

Cancer

SRp20 has been linked to cancer in many instances, as have other alternative splicing proteins. SRp20 has been seen to activate the alternative splicing of the CD44 adhesion molecule by facilitating the splicing of exon v9, which is important for CD44 functionality. Loss of function of SRp20 leading to loss of function of CD44 will lead to loss of “stickiness” of cells, allowing cancer cells to spread to other areas of the body. SRp20 can also affect the alternative splicing of oncogenes and tumor suppressors. Expression of the signaling pathway for SRp20 translation is directly triggered by oncogenic signaling pathways. For example, the Wnt pathway, that is associated with cancer development, also drives further expression of SRp20 and other SR proteins^[8]. These proteins aid in cell growth and proliferation, by increasing expression of proteins that are involved in splicing. One study found that silencing SRp20 slowed cell proliferation, further supporting the idea that SRp20 aids in the process^[1]. Cell cycle regulator proteins FoxM1, Cdc25B, and PLK1 regulate the G2/M cycles and are alternatively spliced by SRp20. These proteins are also associated with regulation of cell apoptosis. When these genes are overexpressed due to overexpressed SRp20, cells will no longer be able to apoptose when they are damaged, and the cell cycle will continue without regulation, causing overactive cell proliferation and eventually cancer. SRp20 has been seen to be overexpressed in ovarian, lung, breast, stomach, skin, bladder, colon, liver, thyroid, and kidney cancer tissue^[9]. SRp20 also regulates genes associated with cellular senescence, or cellular immortality, which contributes to cancer formation. Specifically, SRp20 alternatively splices the TP53 gene, which generates the p53 senescence protein, promoting "cell immortality," which is characteristic of cancer.

Neurological Disorders

Along with cancer, SRp20 mutations have been linked to Alzheimers, a neurodegenerative disorder. SRp20 is involved in alternative splicing of a wide array of RNAs, including that of the TRKB gene which generates TrkB-Shc transcripts that are involved in generation of the disorder. SRp20 also promotes exclusion of exon 10 in the TAU gene, a gene important in establishing microtubules in axons or transport processes. Underexpression of SRp20 results in dysfunction of the TAU gene, less microtubule functionality, and the brain deterioration characteristic of Alzheimers^[10][8].

Other Genetic Disorders

SRp20 and other SR proteins have been shown to prevent R-loops from forming, triple-stranded nucleic acid structures consisting of RNA and DNA. R-loops have been known to promote mutations, recombination, and chromosome rearrangement. One proposed mechanism for R-loop prevention by SRp20 is that SRp20, being a protein involved in RNA metabolism, is a binding partner of the TOP1 protein. Underexpression of TOP1 also promotes R-loop formation. TOP1 has kinase activity that potentially phosphorylates the SR domain of SRp20, which contributes to its function. Underexpression of TOP1 would lead to loss-of-function of SRp20, which could lead to cancer and Alzheimers as mentioned above, as well as cause R-loops to form. R-loops have been associated with disorders such as Down Syndrome^[11].

References

1. ↑ ^{1.0 1.1 1.2 1.3 1.4 1.5} Corbo C, Orru S, Salvatore F. SRp20: an overview of its role in human diseases. Biochem Biophys Res Commun. 2013 Jun 21;436(1):1-5. doi:, 10.1016/j.bbrc.2013.05.027. Epub 2013 May 16. PMID:23685143 doi:http://dx.doi.org/10.1016/j.bbrc.2013.05.027
2. ↑ ^{2.0 2.1} Zahler AM, Lane WS, Stolk JA, Roth MB. SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev. 1992 May;6(5):837-47. PMID:1577277
3. ↑ Ayane M, Preuss U, Kohler G, Nielsen PJ. A differentially expressed murine RNA encoding a protein with similarities to two types of nucleic acid binding motifs. Nucleic Acids Res. 1991 Mar 25;19(6):1273-8. PMID:2030943
4. ↑ Caceres JF, Kornblihtt AR. Alternative splicing: multiple control mechanisms and involvement in human disease. Trends Genet. 2002 Apr;18(4):186-93. PMID:11932019
5. ↑ ^{5.0 5.1 5.2 5.3 5.4} Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Golovanov AP, Stevenin J, Lian LY, Wilson SA, Allain FH. Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8. EMBO J. 2006 Nov 1;25(21):5126-37. Epub 2006 Oct 12. PMID:17036044
6. ↑ ^{6.0 6.1} Shepard PJ, Hertel KJ. The SR protein family. Genome Biol. 2009;10(10):242. doi: 10.1186/gb-2009-10-10-242. Epub 2009 Oct 27. PMID:19857271 doi:http://dx.doi.org/10.1186/gb-2009-10-10-242
7. ↑ ^{7.0 7.1} Huang Y, Gattoni R, Stevenin J, Steitz JA. SR splicing factors serve as adapter proteins for TAP-dependent mRNA export. Mol Cell. 2003 Mar;11(3):837-43. PMID:12667464
8. ↑ ^{8.0 8.1} Corbo C, Orru S, Gemei M, Noto RD, Mirabelli P, Imperlini E, Ruoppolo M, Vecchio LD, Salvatore F. Protein cross-talk in CD133+ colon cancer cells indicates activation of the Wnt pathway and upregulation of SRp20 that is potentially involved in tumorigenicity. Proteomics. 2012 Jun;12(12):2045-59. doi: 10.1002/pmic.201100370. PMID:22623141 doi:http://dx.doi.org/10.1002/pmic.201100370
9. ↑ Jia R, Li C, McCoy JP, Deng CX, Zheng ZM. SRp20 is a proto-oncogene critical for cell proliferation and tumor induction and maintenance. Int J Biol Sci. 2010 Dec 15;6(7):806-26. PMID:21179588
10. ↑ Ebneth A, Godemann R, Stamer K, Illenberger S, Trinczek B, Mandelkow E. Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer's disease. J Cell Biol. 1998 Nov 2;143(3):777-94. PMID:9813097
11. ↑ Naro C, Bielli P, Pagliarini V, Sette C. The interplay between DNA damage response and RNA processing: the unexpected role of splicing factors as gatekeepers of genome stability. Front Genet. 2015 Apr 15;6:142. doi: 10.3389/fgene.2015.00142. eCollection 2015. PMID:25926848 doi:http://dx.doi.org/10.3389/fgene.2015.00142