Srp20-Human Alternative Splicing Factor: Difference between revisions

The SRp20 RRM (aa 1-86) contains a βαββαβ <scene name='78/786034/Imager1/2'>pattern</scene>, 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 <scene name='78/786034/Imager2/2'>stack</scene> 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 <scene name='78/786034/Imager3/1'>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 previously observed only for guanine in the 2 position. U3 <scene name='78/782597/Imager8/4'>Stacks</scene> onto F48 in β3, as W40 and A42 in β2.  However, when bound, U3 <scene name='78/786034/Imager9/1'>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 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 <scene name='78/782597/Imager10/4'>hydrogen bonds</scene>: from the C1 amino protons to Leu80 backbone carbonyl oxygen and to Glu79 side-chain carbonyl oxygen, from C1 N3 to Asn82 amide, and from C1 O2 to Ser81 side chain hydroxyl group.  

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 <scene name='78/786034/Rrmredgreen/1'>SRp20 RRM</scene> 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 <scene name='78/786034/Arginine1/1'>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, but it also does not depend on interaction with the host RRM to retain function<ref name="Hargous">PMID:17036044</ref>.