In vivo expression of the nucleolar group I intron-encoded I-dirI homing endonuclease involves the removal of a spliceosomal intron.
The Didymium iridis DiSSU1 intron is located in the nuclear SSU rDNA and has an unusual twin-ribozyme organization. One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation. The other ribozyme (DiGIR1), which along with the endonuclease-encoding I-DirI open reading frame (ORF) is inserted in DiGIR2, carries out hydrolysis at internal processing sites (IPS1 and IPS2) located at its 3' end. Examination of the in vivo expression of DiSSU1 shows that after excision, DiSSU1 is matured further into the I-DirI mRNA by internal DiGIR1-catalysed cleavage upstream of the ORF 5' end, as well as truncation and polyadenylation downstream of the ORF 3' end. A spliceosomal intron, the first to be reported within a group I intron and the rDNA, is removed before the I-DirI mRNA associates with the polysomes. Taken together, our results imply that DiSSU1 uses a unique combination of intron-supplied ribozyme activity and adaptation to the general RNA polymerase II pathway of mRNA expression to allow a protein to be produced from the RNA polymerase I-transcribed rDNA. (+info
Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA.
Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism. (+info
The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5' splice site.
A U5 snRNP protein, hPrp8, forms a UV-induced crosslink with the 5' splice site (5'SS) RNA within splicing complex B assembled in trans- as well as in cis-splicing reactions. Both yeast and human Prp8 interact with the 5'SS, branch site, polypyrimidine tract, and 3'SS during splicing. To begin to define functional domains in Prp8 we have mapped the site of the 5'SS crosslink within the hPrp8 protein. Immunoprecipitation analysis limited the site of crosslink to the C-terminal 5060-kDa segment of hPrp8. In addition, size comparison of the crosslink-containing peptides generated with different proteolytic reagents with the pattern of fragments predicted from the hPrp8 sequence allowed for mapping of the crosslink to a stretch of five amino acids in the C-terminal portion of hPrp8 (positions 1894-1898). The site of the 5'SS:hPrp8 crosslink falls within a segment spanning the previously defined polypyrimidine tract recognition domain in yPrp8, suggesting that an overlapping region of Prp8 may be involved both in the 5'SS and polypyrimidine tract recognition events. In the context of other known interactions of Prp8, these results suggest that this protein may participate in formation of the catalytic center of the spliceosome. (+info
Genome-wide bioinformatic and molecular analysis of introns in Saccharomyces cerevisiae.
Introns have typically been discovered in an ad hoc fashion: introns are found as a gene is characterized for other reasons. As complete eukaryotic genome sequences become available, better methods for predicting RNA processing signals in raw sequence will be necessary in order to discover genes and predict their expression. Here we present a catalog of 228 yeast introns, arrived at through a combination of bioinformatic and molecular analysis. Introns annotated in the Saccharomyces Genome Database (SGD) were evaluated, questionable introns were removed after failing a test for splicing in vivo, and known introns absent from the SGD annotation were added. A novel branchpoint sequence, AAUUAAC, was identified within an annotated intron that lacks a six-of-seven match to the highly conserved branchpoint consensus UACUAAC. Analysis of the database corroborates many conclusions about pre-mRNA substrate requirements for splicing derived from experimental studies, but indicates that splicing in yeast may not be as rigidly determined by splice-site conservation as had previously been thought. Using this database and a molecular technique that directly displays the lariat intron products of spliced transcripts (intron display), we suggest that the current set of 228 introns is still not complete, and that additional intron-containing genes remain to be discovered in yeast. The database can be accessed at http://www.cse.ucsc.edu/research/compbi o/yeast_introns.html. (+info
An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p.
Pre-mRNA splicing requires dramatic RNA rearrangements hypothesized to be catalyzed by ATP-dependent RNA unwindases of the DExD/H box family. In a rearrangement critical for the fidelity of 5' splice site recognition, a base-pairing interaction between the 5' splice site and U1 snRNA must be switched for a mutually exclusive interaction between the 5' splice site and U6 snRNA. By lengthening the U1:5' splice site duplex, we impeded this switch in a temperature-dependent manner and prevented formation of the spliceosome's catalytic core. Using genetics, we identified the DExD/H box protein Prp28p as a potential mediator of the switch. In vitro, the switch requires both Prp28p and ATP. We propose that Prp28p directs isomerization of RNA at the 5' splice site and promotes fidelity in splicing. (+info
Splicing factor Prp8 governs U4/U6 RNA unwinding during activation of the spliceosome.
The pre-mRNA 5' splice site is recognized by the ACAGA box of U6 spliceosomal RNA prior to catalysis of splicing. We previously identified a mutant U4 spliceosomal RNA, U4-cs1, that masks the ACAGA box in the U4/U6 complex, thus conferring a cold-sensitive splicing phenotype in vivo. Here, we show that U4-cs1 blocks in vitro splicing in a temperature-dependent, reversible manner. Analysis of splicing complexes that accumulate at low temperature shows that U4-cs1 prevents U4/U6 unwinding, an essential step in spliceosome activation. A novel mutation in the evolutionarily conserved U5 snRNP protein Prp8 suppresses the U4-cs1 growth defect. We propose that wild-type Prp8 triggers unwinding of U4 and U6 RNAs only after structurally correct recognition of the 5' splice site by the U6 ACAGA box and that the mutation (prp8-201) relaxes control of unwinding. (+info
Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs.
The U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) involved in pre-mRNA splicing contain seven Sm proteins (B/B', D1, D2, D3, E, F, and G) in common, which assemble around the Sm site present in four of the major spliceosomal small nuclear RNAs (snRNAs). These proteins share a common sequence motif in two segments, Sm1 and Sm2, separated by a short variable linker. Crystal structures of two Sm protein complexes, D3B and D1D2, show that these proteins have a common fold containing an N-terminal helix followed by a strongly bent five-stranded antiparallel beta sheet, and the D1D2 and D3B dimers superpose closely in their core regions, including the dimer interfaces. The crystal structures suggest that the seven Sm proteins could form a closed ring and the snRNAs may be bound in the positively charged central hole. (+info
Processing of endogenous pre-mRNAs in association with SC-35 domains is gene specific.
Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript "trees" of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional "tracks" of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism. (+info