User:Mark Macbeth/Sandbox4: Difference between revisions

= SRp20 is the coolest thing evah! =

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<ref name="Corbo2013">PMID:23685143</ref>. SRp20 has even been found to serve as a splicing factor for its own mRNA, influencing the inclusion of exon 4<ref name="Corbo2013">PMID:23685143</ref>. Another function of SRp20 is its role in export of mRNA out of the nucleus, notably [https://en.wikipedia.org/wiki/Histone_H2A H2A histone] mRNA export<ref name="Hargous">PMID:17036044</ref>.

Attempts to determine the structure of native SRp20 have 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 <scene name='78/782597/Imager0/2'>IgG binding domain</scene> 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 [https://en.wikipedia.org/wiki/Nuclear_magnetic_resonance 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<ref name="Hargous">PMID:17036044</ref>.

The splicing mechanism for SRp20 follows the normal eukaryotic mechanism, in which five different [https://en.wikipedia.org/wiki/SnRNP small nuclear ribonucleoproteins] (snRNPs) bring the splice sites together in order to start the reaction (Figure 1). Specifically, SRp20 and other SR proteins interact with the RNA ligand at the [https://en.wikipedia.org/wiki/Exonic_splicing_enhancer 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 2)<ref name="Shepard">PMID:19857271</ref>. [[Image:mechanism1.png|300px|left|thumb|'''Figure 1.''' Splicing mechanism for eukaryotes. Free 3’OH nucleophile of adenosine in intron attacks phosphorus of phosphate creating a ring structure intron known as a lariat. The free 2’OH in the 5’ splice site (red) acts as the nucleophile to attack the phosphate of the first nucleotide in the 3’ splice site (red) to release the [https://news.brown.edu/articles/2012/06/lariats lariat] intron. The products include the final modified RNA sequence and the lariat which will be recycled.]] [[Image:prettymechanism.png|260px|right|thumb|'''Figure 2.''' SRp20 works with [https://en.wikipedia.org/wiki/U2_spliceosomal_RNA U2] snRNP: Five snRNPs are needed in the eukaryotic splicing mechanism to facilitate the reaction. The U2 snRNP must attach to the 3’ splice site to bring together the 5’ and 3’ splice sites (red). SRp20 facilitates binding of U2 to the 3’ splice site by binding to the exonic splicing enhancer sequence (blue) in the RNA at the backbone. U2 must bind before the other snRNPs can bind to continue the mechanism.]]

The SRP20 RRM (aa 1-86) a βαββαβ <scene name='78/782597/Imager1/3'>pattern</scene>, common of many other RRMs3. For substrate binding, researchers used a 4 base RNA with sequence CAUC, which matches the SRP20 recognition sequence found on corresponding H2A mRNA. The RNA bases each <scene name='78/782597/Imager2/5'>stack</scene> onto an aromatic side chain protruding from one of the SRP20 β-sheets, forming the primary interactions which allow substrate binding to the protein. In particular, C1 <scene name='78/782597/Imager3/2'>stacks</scene> on Y13 in β1, <scene name='78/782597/Imager4/2'>A2</scene> 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 <scene name='78/782597/Imager5/3'>syn</scene> conformation when bound to the RRM, something that was observed only for guanine in the 2 position previously. U3 <scene name='78/782597/Imager8/4'>Stacks</scene> onto F48 in β3, also W40 and A42 in β2,  however when bound, U3 <scene name='78/782597/Imager9/2'>bulges</scene> out of line in comparison to the rest of the substrate. C4 partially stacks over <scene name='78/782597/Imager6/4'>A2</scene>, and also forms hydrogen <scene name='78/782597/Imager7/3'>bonds</scene> 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 substrate sequence does not significantly impact binding affinity, while 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 nonspecifically3. The Srp20 RRM is able to recognize C1 with high specificity primarily through 4 <scene name='78/782597/Imager10/4'>hydrogen bonds</scene>: from the C1 amino protons to Leu 80 backbone carbonyl oxygen and to Glu 79 side-chain carbonyl oxygen, from C1 N3 to Asn82 amide, and C1 O2 with Ser 81 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 more diverse range of substrates, or even original substrates that were mutated during replication (eg. H2A mRNA with a point mutation) thereby increasing organism survival chance by reducing the probability of physiological impact as a result of certain mutations<ref name="Hargous">PMID:17036044</ref>.  

[[Image:AASequence.jpg|350px|right|thumb|'''Figure 3.''' SRp20 Domain Representation. Shown are the RRM (green), the TAP binding linker (red line) and SR-rich domain (blue).]]

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 translation3. In particular SRp20 promotes the export of H2A histone mRNA, by binding the CAUC consensus sequence on the mRNA and binding TAP. Previous experiments have shown that Srp20 binding TAP is dependent on presence of both the <scene name='78/782597/Rrmredgreen/1'>SRp20 RRM</scene> and a short arginine rich C-terminal segment after the RRM (aa 1-83 and 84-90 respectively) ('''Figure 3'''). Previous research also shows that mutation of any one of the three arginine residues present 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 <scene name='78/782597/Arginine1/4'>first arginine residue</scene> 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, it does not depend on interaction with the host RRM to retain function<ref name="Hargous">PMID:17036044</ref>.