Prp24: Difference between revisions

Prp24 contains four RRMs: RRM 1, RRM 2, RRM 3, and RRM 4.  These motifs have a <scene name='Sandbox_Reserved_340/2ghp/19'>canonical structure of a platform of four β-strands with two α-helices on one side of the β-sheet plane</scene>.  These RRMs are present in many proteins that bind to single stranded regions of RNA <ref name="RRM"/> and their presence in Prp24 supports a role for the annealing of U4 and U6 snRNAs into the U4/U6 structure.

Within each RRM there are RNP consensus domains , which are the regions in the β-strands thought to actually interact with the RNA <ref name="RRM">PMID:15853797</ref>.  These regions seem to be very important in Prp24 and its interaction with U4 and U6.  The study that first identified a probable link between the Prp24 protein and U4/U6 found <scene name='Sandbox_Reserved_340/2ghp/28'>mutations in RNP 1 and RNP 2 of the RRM 3</scene> that rescued a cold-sensitive phenotype caused by a U4 mutation in stem II of U4/U6 <ref name="Shannon"/>.  Two further studies <ref name="Vidaver"/> <ref name="Kwan">PMID:15811912</ref> showed that the mutation of <scene name='Sandbox_Reserved_340/2ghp/29'>three highly conserved residues in the RNP domains</scene> of any of the four RRMs conferred either temperature-sensitive growth or lethality to yeast cells.  

Prp24 is part of the normal U6 snRNP, along with seven [http://en.wikipedia.org/wiki/Lsm Lsm proteins] <ref name="Brow review"/>.  After interaction with the U4 snRNP to form the U4/U6 di-snRNP, Prp24 departs from the di-snRNP before the addition of U5 to form the tri-snRNP U4/U6.U5 <ref name="Brow review"/>.  Through many studies, it has been shown that Prp24 interacts extensively with specific sites on U6, as well with the Lsm protein ring on the 3' terminal end of the U6 snRNA.

Prp24 co-immunoprecipitates with free U6 and U4/U6 di-snRNP, indicating that it is closely associated with these structures <ref name="Shannon"/><ref name="Ghetti">PMID:7585243</ref>.  Initial investigation revealed that Prp24 binds within the 30-56 nucleotide region of free U6, as well as to stem II of U4/U6 in the 39-56 and 67-70 nucleotide regions of U6 <ref name="Ghetti"/>.  Further investigation of the structure showed that Prp24 very likely binds directly to the 40-43 nucleotides of U6 based on chemical modification of naked U6 snRNA compared to free U6 snRNP<ref name="Jandrositz"/>.   

The main function of Prp24 seems to be directly related to formation of the U4/U6 complex, particularly based on the evidence that Prp24 is present in U6 and U4/U6, but not U4/U6.U5 <ref name="Shannon"/><ref name="Ghetti"/><ref name="Jandrositz"/>.  Prp24 greatly increases the rate and efficiency of U4/U6 annealing <ref name="Raghunathan">PMID:9452384</ref> and mutations in Prp24 have been shown to prevent the formation of the U4/U6 di-snRNP <ref name="Lygerou">PMID:10022888</ref>.  Although the exact mechanism by which Prp24 promotes annealing of U4 and U6 is not known, it has been suggested that Prp24 may stabilize the secondary structure of U6 to allow it to interact with U4 in order to allow formation of U4/U6 <ref name="Vidaver"/>.

The interactions of Prp24 with U6 and U4/U6 are very important in proper spliceosome assembly, as evidenced by mutations in Prp24 that suppress mutations in U6 or U4.  Mutations in RRM 2 and RRM 3 suppress the effects of mutations in the 3' stem of U6, in a region termed the telestem <ref name="Vidaver"/>.  A later study showed that RRM 1 binds with high affinity to U6 and is important in interactions with mutations in the 3' region of U6 <ref name="Kwan"/>.  Mutations in two RNP consensus domains of Prp24 suppress the effects of mutations in U4 in the stem II region of U4/U6 <ref name="Shannon"/>.  Truncation and deletion of internal segments of U6 showed that the central region of U6 is the most important region for interaction with Prp24 and that RRM 1 and RRM 2 are the likely portions of Prp24 that interact with U6 in this region <ref name="Kwan"/>.  Deletion of conserved residues in the C-terminal domain of Prp24 caused temperature sensitive growth and reduced levels of U4/U6, suggesting that this region, containing the fourth RRM, is also important for interactions in assembly of the spliceosome <ref name="Rader"/>.

NMR analysis of fragments of U6 sequence and regions of Prp24 have further defined and supported the interactions of the protein with U6 snRNA.  Analysis of an RNA oligonucleotide containing sequences identical to nucleotides 41-46 and 83-88 of U6 (the regions proposed to bind to Prp24) with a truncated protein containing RRMs 1 and 2 of Prp24 showed that these RRMs interacted sequence specifically with these nucleotides <ref name="Bae"/>.  Another NMR study identified sequence specific interaction of <scene name='Sandbox_Reserved_340/2kh9/3'>RRM 2 with RNA</scene>, and it was determined from this that the most likely region of U6 for interaction was the AGAGAU sequence of nucleotides 49-54, within the region of the previously predicted interaction <ref name="Martin">PMID:20181740</ref>.  From this, it was predicted that RRM 1 would interact with the GAUCAG sequence of nucleotides 55-60 <ref name="Martin"/>.   

The Lsm proteins are a set of seven proteins found in the U6 snRNP that form a ring around the 3' end of U6 <ref name="Karaduman2008"/>.  An interaction between Prp24 and the Lsm proteins was suggested upon identification of the Lsms and the fact that formation of U4/U6 by Prp24 was less efficient in deproteinized solutions of U4 and U6 snRNAs <ref name="Raghunathan"/>, as well as the identification of a genetic interaction between the genes for Prp24 and Lsm4 <ref name="Mayes">DOI:10.1093/emboj/18.15.4321</ref>.  This proposed interaction was further supported by UV cross-linking experiments in which both Prp24 and Lsm4 were immunoprecipitated with U6 after cross-linking <ref name="Vidal">PMID:10580475</ref>.  It has also been shown that both Prp24 and Lsm4, as well as the other Lsm proteins, require the 3' end of U6 in order to interact with the snRNA <ref name="Vidal"/>, suggesting that this may be the region where the both Lsm proteins interact with U6 and Prp24.  Genome-wide protein interaction screens showed that Prp24 interacts additionally with Lsm 2, 5, 6, 7 and 8, and that overexpression of either Prp24 or Lsm4 can complement or intensify the effects of a defect in the other protein <ref name="Fromont">PMID:10900456</ref>, further supporting an interaction between the Lsm proteins and Prp24.

Evidence for a direct interaction of Prp24 and the Lsm proteins comes from a study in which the conserved residues in the C-terminal domain of Prp24 were deleted<ref name="Rader"/>.  These deletions resulted in lowered levels of U4/U6 and very little interaction with the Lsm proteins as compared to wild-type Prp24<ref name="Rader"/>.  This indicated that Prp24 interacts directly with the Lsm proteins and the C-terminal domain is necessary for this interaction <ref name="Rader"/>.  Another indication of interaction between Prp24 and the Lsm proteins stems from a study showing that Lsm6 and Lsm7 are necessary in cells that require the recycling of the U4/U6 complex for splicing and that the presence of these two proteins increases the efficiency of annealing of U4 and U6 <ref name="Verdone">PMID:15324666</ref>.  This suggests that Lsm6 and 7 are involved in a necessary interaction with Prp24 in the formation of U4/U6 di-snRNP.   

More recently, a study has suggested that Prp24 interacts specifically with all the Lsm proteins involved in the U6 snRNP, and that the proteins act together as molecular chaperones to restructure and stabilize the 3' stem of U6 for base-pairing with U4 in stem II of U4/U6<ref name="Karaduman2006">PMID:16410014</ref>.  Further support for specific interactions of Prp24 with the Lsm proteins comes from an electron microscopy study that showed Prp24 at specific distances from the subunits of the Lsm ring, suggesting that it interacts from a specified position within the U6 snRNP <ref name="Karaduman2006"/>.

Several additional roles for Prp24 have been suggested in spliceosome assembly/disassembly, although nothing has been sufficiently supported.  A genetic interaction in which Prp24 mutation suppressed a Prp21 (a component of the U2 snRNP) mutation suggested that the two proteins may interact during the base pairing of U2 and U6 at the 5' splice site; however, further investigation failed to produce evidence of a definite interaction between the two proteins, although the authors maintained that a transient interaction between Prp24 and Prp21 may exist in an intermediate form of the assembling spliceosome <ref name="Vaidya"/>.  It has also been suggested that Prp24 may also assist in the formation of U4/U5.U6 <ref name="Ryan">PMID:12212846</ref> or in the destabilization of U6 from U2 upon completion of splicing to release free U6 snRNP,<ref name="Vidaver"/>, but there is insufficient evidence to conclusively support these functions of Prp24.