Prp24

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Template:STRUCTURE 2ghp

Prp24 (Pre-mRNA splicing Protein 24) is a Saccharomyces cerevisiae yeast protein that functions in the formation of base pair interactions between the U6 and U4 snRNPs (small nuclear ribonucleoproteins) to form the U4/U6 di-snRNP during the assembly of the spliceosome . This protein contains four RNA recognition motifs (RRMs) that function in the binding of Prp24 to U6 snRNA. These RRMs domains are conserved in structure and sequence in proteins orthologous to Prp24 in Homo sapiens and Schizosaccharomyces pombe, as well as in other proteins containing RRMs.

IntroductionIntroduction

Pre-mRNA SplicingPre-mRNA Splicing

Pre-mRNA splicing is an essential process in eukaryotes that removes non-coding introns from a pre-mRNA transcript and splices coding exons together before the mRNA is exported from the nucleus for translation into a protein. Splicing requires five snRNPs (U1, U2, U4, U5, U6), several other proteins, and the input of energy from ATP. The U1 and U2 snRNPs assemble individually on the pre-mRNA transcript, while U4 and U6 form a U4/U6 di-snRNP before interacting with U5 to form a U4/U6.U5 tri-snRNP that combines with U1 and U2 at the pre-mRNA transcript. U4 and U1 then depart, and after conformational changes and base pair formation with the pre-mRNA the remaining snRNPs form the catalytically active spliceosome. Two transesterification reactions then occur; the first reaction is the nucleophillic attack of the a phosphate group at the end of the 5' exon by the 2' hydroxyl of a specific adenosine at the branch point sequence of the intron. This is then followed by the nucleophillic attack of the phosphorous group linking the 3' exon to the intron by the 3' hydroxyl of the 5' exon. These splicing reactions, in addition to the addition of 7-methylguanosine 5' cap and a 3' polyadenosine tail, results in a mature mRNA transcript that can be exported from the nucleus and translated into protein.

U6 and U4 snRNPsU6 and U4 snRNPs

U6 is considered to be one of the most catalytically important snRNAs in the spliceosome, as it interacts directly with the 5' splice site through base pairing. It is thought to undergoe three conformational changes throughout the entire process of splicing and splicesome assembly; it exists as one conformation as free U6 snRNP, another conformation as part of the U4/U6 di-snRNP and a third conformation associated with U2 and the pre-mRNA. In addition to Prp24, U6 is associated with seven other proteins, Lsm 2-8, which form a ring around the 3' portion of the U6 snRNA (reference Brow 2002).

The U4 snRNA is though to be non-catalytic because it leaves the spliceosome before the transesterification reactions occur. Its function is instead thought to be aiding U6 in maintaining a conformation that will enable it to interact with U2 and the 5' splice site. The U4 snRNP contains the U4 snRNA, a ring complex of the Sm proteins B-G, and the proteins Prp3, Prp4, and Snu13 (reference Brow 2002).

The annealing of the U4 and U6 snRNAs to form the U4/U6 di-snRNP complex, which contains the additional proteins Prp6 and Prp31, is an essential process in the formation of the spliceosome. It enables interaction with the U5 snRNP to form the U4/U6.U5 complex which serves to deliver the U5 and U6 snRNPs to the appropriate sites in the pre-spliceosome complex to form a catalytically active spliceosome upon departure of U1 and U4.

Role of Prp24 in SplicingRole of Prp24 in Splicing

Prp24 is a U6 snNRP protein that functions in the annealing of the U4 and U6 snRNPs during the assembly of the spliceosome. This protein was first identified in a genetic screen as mutated gene that caused an accumulation of pre-mRNA (reference Vijayraghavan et al. 1989). It's first functional role was suggested after several mutant forms of the protein were found to suppress a cold sensitive growth defect caused by mutations in the U4 snRNA (reference Shannon and Guthrie 1991). It is thought that Prp24 helps to stabilize the U6 snRNA and hold it in a conformation that promotes base pairing interactions with the U4 snRNA to form the stem I and stem II structures of the U4/U6 di-snRNP. Prp24 departs from the U4/U6 complex before the formation of the U4/U6.U5 tri-snRNP, but it has been suggested that Prp24 may also play a role in the dissociation of U4 from U6 during the base pairing of U6 with U2 and the 5' splice site. This additional role for Prp24, however, has not been experimentally supported.

StructureStructure

spinqualitylabelsall models X-ray crystallographic structure of the first three RRMs of Prp24 Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

The key structural elements of Prp24 are the conserved RNA recognition motifs (RRMs). These motifs are found in many proteins with RNA binding properties and are contain conserved RNP elements that are recognizable from their primary sequence. For several years, Prp24 was thought to contain three RRMs, termed RRM 1, RRM 2, and RRM 3 (green link)(reference Shannon and Guthrie 1991). However, analysis of homologs of Prp24 from several different species allowed the identification of a fourth RRM in Prp24 of S. cerevisiae, albeit one that was much less highly conserved and not easily recognizable by its RNP-consensus domain (reference Rader and Guthrie 2002).

RNA Recognition MotifsRNA Recognition Motifs

Prp24 contains four RRMs, RRM 1, RRM 2, RRM 3, and RRM 4. These motifs have a canonical structure of a platform of four β-strands with two α-helices on one side of the β-sheet plane (green link). These RRMs are present in many proteins that bind to to single stranded regions of RNA (reference!!!!) 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 two RNP consensus domains. These are the regions in the β-strands that are thought to actually interact with the RNA. 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 that mutations in RNP 1 and RNP 2 of the carboxy terminal RRM (green link) rescued a cold-sensitive phenotype caused by a U4 mutation in stem II of U4/U6 (Shannon and Guthrie 1991). Two further studies (reference Vidaver et al. 1999 and Kwan and Brow 2005) showed that the mutation of three highly conserved residues in the RNP domains of any of the four RRMs (green link) conferred either temperature-sensitive growth or lethality to yeast cells.

Structural ModelsStructural Models

A recent study (reference Bae et al. 2008) examined the structure of the N-terminal domain and first three RRMs of Prp24 by X-ray crystallography (green link back to main structure?). The resultant structure showed Prp24 as an octameric protein, consisting of eight chains arranged in two nearly symmetrical tetramers. This was the first suggested that Prp24 functioned as an multimer, so it is unclear what the significance of this result is (reference Bae et al. 2008). Their crystal structure also showed extensive interactions between both RRM 1 and 2 (green link) and RRM 2 and 3 (green link), and NMR analysis of protein fragments containing either RRMs 1 and 2 or RRMs 2 and 3 showed that these interactions existed in solution as well. Interestingly, these interactions seemed to block the proposed U6 binding sites of RRM 1 and 2; NMR analysis of the RRM 1 and 2 protein fragment with an RNA oligomer containing the U6 sequences thought to bind the RRMs showed a largely canonical interaction of the RNA with the RRMs, suggesting that Prp24 may undergo conformational changes in the binding of U6.

Functional InteractionsFunctional Interactions

spinqualitylabelsall models Insert caption here Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Prp24 is part of the normal U6 snRNP, along with seven Lsm proteins. 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 (reference!!). 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.

U6 snRNA and U4/U6 snRNPU6 snRNA and U4/U6 snRNP

U6 is arguably the most structurally dynamic snRNA involved in the splicing process, seemingly undergoing at least three different structures: it exists in one conformation as free U6 snRNP, another when base paired in the U4/U6 di-snRNP, and a third in base pairing interactions with U2 and the 5' splice site. One large role suggested for Prp24 has been in assisting in the conformational changes between the free U6 structure and the U4/U6 conformation.

Prp24 coimmunoprecipitates with free U6 and U4/U6 di-snRNP, indicating that it is closely associated with these structures (reference Shannon and Guthrie 1991, Ghetti et al. 1995). 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 (reference Ghetti et al. 1995). 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 (reference Jandrositz and Guthrie 1995).

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 (reference Shannon and Guthrie 1991, Ghetti et al. 1995, and Jandrositz and Guthrei 1995). Prp24 greatly increases the rate and efficiency of U4/U6 annealing (Raghunathan and Guthrie 1998) and mutations in Prp24 have been shown to prevent the formation of the U4/U6 di-snRNP (Lygerou et al. 1999). Although the exact mechanism by which Prp24 promotes annealing of U4 and U6, 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 (reference Vidaver et al. 1999)

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 (reference Vidaver et al. 1999) (the existence of the telestem has been challenged by recent U6 secondary structure models (reference Karaduman et al. 2008 and Dunn and Rader 2010)). 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 (reference Kwan and Brow 2005). Mutations in two RNP consensus domains of Prp24 suppress the effects of mutations in U4 in the stem II region of U4/U6 (reference Shannon and Guthrie 1991). 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 (reference Kwan and Brow 2005). 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 too, containing the fourth RRM, is important for interactions in assembly of the spliceosome (Rader and Guthrie 2002).

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 (Bae et al. 2007). Another NMR study identified sequence specific interaction of RRM 2 with RNA, and it was determined from this that most likely region of U6 for interaction was the AGAGAU sequence of nucleotides 49-54, within the region of previously predicted interaction (Martin-Tumasz et al. 2010). From this, it was predicted that RRM 1 would interact with the GAUCAG sequence of nucleotides 55-60 (Martin-Tumasz et al. 2010).

Lsm ProteinsLsm Proteins

The Lsm proteins are a set of seven proteins found in the U6 snRNP that form a ring around the 3' end of U6 (reference Karaduman et al. 2008). 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 (reference Raghunathan and Guthrie), as well as the identification of a genetic interaction between the genes for Prp24 and Lsm4 (Mayes et al. 1999). This proposed interaction was further supported by UV cross-linking experiments in which both Prp24 and Lsm4 were immunoprecipitated with U6 after cross-linking (reference Vidal et al. 1999). 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 (Vidal et al. 1999), suggesting that this may be the region where the proteins interact. 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 (Fromont-Racine et al. 2000), 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, resulting in lowered levels of U4/U6 and very literal interaction with the Lsm proteins as compared to wild-type Prp24, indicating that Prp24 interacts directly with the Lsm proteins and the C-terminal domain is necessary for this interaction (Rader and Guthrie 2002). 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 (Verdone et al. 2004). 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(Karaduman et al. 2006). Further support for specific interactions of Prp24 with the Lsm proteins comes from an electron microscopy study that showed Prp24 at specific differences from the subunits of the Lsm ring, suggesting that it interacts from a specified position within the U6 snRNP (Karaduman et al. 2006).

Additional InteractionsAdditional Interactions

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 (reference Vaidya et al. 1996). However, further investigation failed to produce evidence of an interaction between the two proteins, but the authors maintained that a transient interaction between Prp24 and Prp21 may exist in an intermediate form of the assembling spliceosome (reference Vaidya and Vijayraghavan 1998). It has also been suggested that Prp24 may serve in the destabilization of the U4/U6 complex to allow base pairing of U5 or in destabilization of U6 from U2 upon completion of splicing to release free U6 snRNP, but again, there have been no studies showing support for these roles of the protein.