Introduction

Overview

Structure Determination

Attempts to determine the structure of native SRP20 were 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 bsheets. 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 soluble IgG-binding domain 1 of Streptococcal protein G to the N terminus of the protein, providing the first published structure of the SRP20 RRM via NMR3. 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.

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 (refer to mechanism figure). 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 (refer to the mechanism figure including the snRNPs).

Structural Highlights

RNA Recognition Motif

The SRP20 RRM (aa 1-86) a βαββαβ pattern, 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 stack onto an aromatic side chain protruding from one of the SRP20 b-sheets, forming the primary interactions which allow substrate binding to the protein. In particular, C1 stacks on Y13 in b1, A2 stacks on F50 in b3, and F48 of b3 sits in between the sugar rings of C1 and A2. It should also be noted that A2 adopts an irregular syn conformation when bound to the RRM, something that was observed only for guanine in the 2 position previously3. U3 stacks onto F48 in b3, also W40 and A42 in b2, however when bound, U3 bulges out of line in comparison to the rest of the substrate. C4 partially stacks over A2, and also forms hydrogen bonds between the C4 amino group and A2 2’ 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 hydrogen bonds: 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.

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 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 SRP20RRM and a short arginine rich C-terminal segment after the RRM (aa 1-83 and 84-90 respectively). 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 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.

SR Domain

PG8 and SRP20

Medical Significance