User:Verda S. Mirza/Sandbox 1
From Proteopedia
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All About SRp20
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Introduction
Alternative RNA splicing is a significant post-transcriptional process that allows diversity of gene expression. Initially, a gene is transcribed into pre-messenger RNA (pre-mRNA). The pre-mRNA contains introns, which are sequences that are not translated into protein, and exons, which code for proteins. In alternative RNA splicing, exons are either removed or retained in the mRNA in different combinations, creating diverse arrangements of mRNAs from one pre-mRNA. This process is carried out with splicing factors which are proteins that remove introns and exons via in spliceosomes1.
SRp20
The sequence-specific RNA binding protein SRp20 (gene name SRSF3) is a splicing factor and one of the smallest member of the serine and arginine-rich (SR) protein family that plays a significant role in alternative splicing of exons1,3. It has been found that SRp20 might be involved in other cellular functions as well such as termination of transcription, export of mRNA from the nucleus, RNA translation, and 3' polyadenylation.
Function
SRp20 is a splicing factor involved in regulation of many genes through alternative splicing of exons through interaction with cis-elements of RNA1,10. Additionally, it contains an auto-regulatory activity in which it can alternatively splice its own mRNA through including exon 4 thus reducing the length of its protein11,12,13.
Other Functions
SRp20 has been linked to termination of transcription by either degrading the RNA sequence downstream from the cleavage site or removal of RNA polymerase from the DNA4. Furthermore, SRp20 might play a role in export of mature mRNAs by promoting the recruitment of TAP, which is a receptor for mRNA export out of the nucleus5. Additionally, it has been found that SRp20 and PCBP2, which is a protein that binds to internal ribosome entry site (IRES) RNA sequences in picornavirus, interact with each other to initiate viral translation6. Thus, these findings indicate SRp20 playing a role in protein translation. SRp20 is also suggested to allow 3' terminal exon to be recognized by polyadenylation factors8.
Disease
Cancer
There have been findings that support SRp20 role in cellular proliferation/maturation. It was discovered that there was an overexpression of SRp20 in breast cancer tissues and when SRp20 was reduced in the cancer cells via siRNA targeting SRp20 mRNA, there was reduction in cell proliferation and increase cellular apoptosis7. For example, it was speculated that SRp20 might be involved in alternative splicing of FoxM1, a transcription factor involved in cellular proliferation, by either the inclusion or exclusion of exon 9 in FoxM1 transcript. If exon 9 was excluded from the FoxM1 mRNA via SRp20 then there was an increase in FoxM1 expression, cellular proliferation, and reduction in cell apoptosis7. Furthermore, pyruvate kinase M (PK-M) gene is responsible for aerobic glycolysis and tumor growth. SRp20 allows for the expression of the oncogenic isoform of PK-M through including exon 10 of the PK-M transcript. Due to the expression of the oncogenic isoform of PK-M, this continues to promote aerobic glycolysis and tumor growth9. Due to the alternative splicing functionality of SRp20, it effects many other genes involved in cancer other than the ones discussed above such as CD44 gene, TAU gene, TP53 gene, and involved in WnT signaling pathway1. Although it has been understood the SRp20 plays a crucial role in cancer cells, it is still unclear mechanism of SRp20 in the genes it effects how its structure contributes to the development of oncogenic genes7,14.
Structure
RMM
N-terminal RNA recognition motif β1α1β2β3α2β3 Topology standard for an RRM A four stranded β-sheet and two α-helices Most commonly, three aromatic side-chains (in β-3 and β-1 strands) accommodate two nucleotides. Recognition enables binding of SRp20 to RNA
mRNA Maturation
Structural highlights
Poor Solubility Problem
Protein has poor solubility in its free state This problem was resolved by studying the proteins either: Fusing the RRM (RNA-recognition motif) with the immunoglobulin G-binding domain 1 of Streptococcal protein G GB1 solubility tag Or In a solution containing charged amino acids From there, the structure of the free protein could be determined
RNA Interactions
1H-15N HSQC results showed a large hydrophobic β-sheet on the RRM binds to the RNA with all four bases contacting one of the four aromatic residues” (hydrophobic interactions) Other structural studies show that amino acids of the β-hairpin are directly hydrogen bonded to bases of nucleic acid targets Using a smaller peptide chain reduced the NMR broadening seen with longer peptides (allowing for structure determination), though the binding affinity was also reduced
C1 and A2 stack on Y13 in β1 and F50 in β3 (aromatic side chains), respectively. (IMAGE)
The residue F48 (purple) inserts between the sugar rings of C1 and A2 (IMAGE)
Looking at the ligand
The conformation of U3 and C4 is unusual because U3 bulges out while C4 stacks over A2, partially. (IMAGE or Video showing bulge and stacking)
A2 also adopts an unusual syn conformation (IMAGE)
C4 is maintained in its position by a hydrogen bond between C4 amino group and the A2 2’ oxygen (IMAGE)
RRM Domain Interactions
U3 interacts with Phe 48, Trp 40, Ala 42, and with the β2-3 loop of the RRM (IMAGE)
C1 is recognized definitively by the RRM (IMAGE) C1 amino protons hydrogen bond with Leu 80 carbonyl oxygen and Glu 79 side-chain carbonyl oxygen. C1 N3 hydrogen bonds with Asn 82 amide. C1 O2 hydrogen bonds with Ser 81 hydroxyl group.
Ser-Arg Rich Domain
Specificity
4 nt can be accommodated by RRM β-sheet, but recognition is only partially sequence specific. CAUC C1 more specific, A2 and U3 less specific It is uncertain whether C4 is specifically recognized by the RRM A is prefered over G at the 2 position, but no indication of preference over U or C U3 is even less specific, could be C, G or A The recognition of C1 is functionally necessary because a C to G mutation within the histone mRNA can impair RNA export
Advantages of low specificity
Less evolutionary pressure on bound RNA (ideal for exonic sequences) More RNA sequences can be targeted SRp20 can associate/disassociate with RNA more easily Important for highly dynamic RNA metabolism processes RNA binding affinity can be modulated by protein-protein interactions (which are dependant on the level of phosphorylation) This can be used to tune post-transcriptional gene expression
Relationship to 9G8
SRp20 and 9G8 are both sequence specific RNA binding proteins. They are the smallest members of the Serine-and-Arginine Rich (SR) protein family. Both RNA Recognition Motifs (RRMs) have a similar βαββαβ topology. SRp20 and 9G8 are 80% identical. The sequence alignment shows the alignment of the RRMs of SRp20 and 9G8 (Hargous et al., 2006). SRp20 binds pyrimidine rich areas while 9G8 binds purine rich areas.This difference in binding comes from the fact that 9G8 has a zinc knuckle that recognizes GAC triplets (Cavaloc et al., 1999). 9G8s RRM is followed by a zinc knuckle and then the SR domain whereas SRp20s RRM is followed directly by the SR domain. When 9G8 lacks a zinc knuckle, it binds pyrimidine-rich sequences like SRp20 (Hargous et al., 2006). The zinc knuckle of 9G8 contains glycine residues at positions 5 and 8 and charged residues at positions 6 and 13 that are highly conserved (Cavaloc et al., 1999). Due to the poor solubility problem, a structure for the zinc knuckle of 9G8 is not available to show in an image. A study by Huang and Steitz showed that 9G8 and SRp20 promote the export of mRNA from the nucleus. There is a 101-nt sequence in the coding region of mouse histone H2a mRNA that promotes the the export of intronless human β-globin cDNA (LINK!!! https://en.wikipedia.org/wiki/Complementary_DNA) transcripts (Huang and Carmichael, 1997). Of this 101-nt sequence, there is specifically a 22-nt sequence that is necessary for export activity. When this 101-nt sequence was not present, mRNA was not properly processed, and RNA accumulated in the nucleus; however, when the 101-nt sequence was present mRNA export from the nucleus was improved almost 4-fold. This 101-nt sequence improves export and polyadenylation, but removing the 22-nt sequence stopped both of these activities suggesting that the 22-nt sequence is required for successful nuclear export of β-globin cDNA. UV cross-linking (LINK!!! https://en.wikipedia.org/wiki/Cross-link) and immunoprecipitation (LINK!!! https://en.wikipedia.org/wiki/Immunoprecipitation) experiments determined the proteins that specifically associate with the 22-nt sequence. It was determined that SR proteins were associating with the 22-nt sequence by adding antibodies specific for SRp20 then 9G8. SRp20 antibodies inhibited mRNA export 3-fold while 9G8 antibodies inhibited mRNA export at least 10-fold showing that SRp20 and 9G8 are active factors that promote mRNA export. It was shown that SRp20 and 9G8 are cross-linked to polyadenylated RNA in the nucleus and cytoplasm showing that both proteins play a direct role in mRNA export from the nucleus (Huang and Steitz, 2001).
Relevance and Conclusions
Understanding and recognizing the mechanisms that SRp20 is involved in can help find treatment and management of cancer patients The use of SR proteins (such as SRp20) may in the future be used for targeted therapy No structure for the C-term domain
Future Directions
Recent structural studies emphasize that not only the β-sheet surface but also the loops connecting β-strands and a-helices can be crucial for nucleic acid recognition → future research looking more closely at this possibility Farther research could be done investigating the process of RS domain phosphorylation and how it controls splicing Look farther into the specificity or lack of specificity relating to SR proteins
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References
Cle’ry A, Blatter M, Allain FHT. 2008. RNA recognition motifs: boring? Not quite. Current Opinion in Structural Biology 18: 290–298. Doi: 10.1016/j.sbi.2008.04.002
Corbo C, Orrù S, Salvatore F. 2013. SRp20: An overview of its role in human diseases. Biochemical and Biophysical Research Communications 436: 1–5. Doi: 10.1016/j.bbrc.2013.05.027
Hargous Y, Hautbergue GM, Tintaru AM, Skrisovska L, Golovanov AP, et. al. 2006. The EMBO Journal ) 25: 5126–5137. Doi: :10.1038/ sj.emboj.7601385
Huang Y, Steitz J A. 2001. Splicing Factors SRp20 and 9G8 Promote the Nucleocytoplasmic Export of mRNA. Molecular Cell 7:889-905. Doi: 10.1016/51097-2765(01)00233-7
