A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family.

Abstract

We have isolated a cold-sensitive mutant of Saccharomyces cerevisiae in which the first step of mRNA splicing is inhibited. The growth and splicing defects are recessive and cosegregate, thus defining a single essential gene (PRP28). The wild-type PRP28 gene was cloned, and sequence analysis reveals extensive homology to a family of proteins that are thought to function as ATP-dependent RNA helicases. The cold sensitivity is caused by a glycine-to-glutamic acid change in a conserved sequence motif. Interestingly, double mutants containing conditional alleles of PRP28 and PRP24, which encodes a U6 snRNA-binding protein, are inviable. In addition, a suppressor of prp28-1 is a mutant allele of PRP8, which encodes a U5 protein, thus linking PRP28 with U5. These data are consistent with a scenario in which PRP28 acts to unwind the U4/U6 base-pairing interaction in the U4/U6/U5 snRNP, facilitating the first covalent step of splicing.

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing

“…Since PRP2 activity is an essential requirement for the first transesterification reaction (27) PRP2A spliceosomes could not process pre-mRNA into spliced mRNA (Fig. 3A, lanes 3-5), and they formed splicing complex I but no splicing complex 11 (28,29 and data not shown). Complementation with a heat-inactivated temperature-sensitive prp8 extract restored splicing activity (Fig.…”

“…A prp8 allele has been isolated as a genetic suppressor ofthe DEAD-box protein splicing factor PRP28, a putative RNA helicase. From this a model was proposed in which PRP28 protein destabilizes the U4/U6 snRNA interaction prior to step 1 of splicing, counterbalanced by a stabilizing effect of PRP8 protein (11). A mutation affecting another DEAD-box protein, DED1, has been isolated as a cold-sensitive suppressor of a temperature-sensitive prp8 allele (12).…”

The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5′ splice site

“…In addition to the above-discussed contacts with the pre-mRNA, Prp8 is expected to interact with a number of spliceosomal components+ An allele of PRP8 ( prp8-201) was found to suppress a cold-sensitive allele of U4 snRNA+ The mutation in prp8-201 maps close to the region involved in polypyrimidine tract recognition (Kuhn et al+, 1999;Umen & Guthrie, 1996)+ Prp40, a component of yeast U1 snRNP, physically interacts with the proline-rich domain at the N-terminus of yPrp8 (Abovich & Rosbash, 1997)+ Finally, Prp8 was shown to genetically interact with two putative helicases: DED1 (Jamieson et al+, 1991) and Prp28 (Strauss & Guthrie, 1991)+ Interestingly, the human homolog of Prp28 (U5-100kD) is also an integral component of the U5 snRNP (Teigelkamp et al+, 1997), suggesting that Prp8 may directly interact with Prp28 and possibly other U5 snRNP components, including U5 snRNA+ While the crosslinking studies have confirmed a close contact between Prp8 and pre-mRNA, Prp8 also has been shown to interact with a series of other spliceosomal components throughout the reaction+ Thus, it is likely that Prp8 plays a central role in splicing, coordinating a series of events through interactions with several spliceosomal components, and perhaps participating in formation of the active site of the splice-osome+ This could be achieved either by providing a binding site for the splice sites, or possibly also by contributing to the chemical reaction in a more direct way+ At present, the mechanistic details concerning the pre-mRNA splicing catalysis are not known+ The catalytic center is likely to involve a combination of specific RNA:RNA interactions and other contacts that rely on amino acid residues+ Because of the discussed close contacts between Prp8, pre-mRNA, and other spliceosomal components, as well as the established RNA: RNA interactions involving U2 and U6 snRNAs, it seems likely that Prp8, along with U2 and U6 snRNAs, participates in formation of the catalytic center+…”

The biochemical defects ofprp4-1andprp6-1yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP

“…However, genetic approaches have led to the isolation of many splicing deficient mutants, called prp mutants (20,21). Some PRP genes encode snRNP components, such as PRP4, PRP8 and PRP24 (14,19,(22)(23)(24) and others, such as PRP2, PRP5, PRP9, PRP11, PRP16, PRP22 and PRP28, are implicated in spliceosome assembly or disassembly (25)(26)(27)(28)(29)(30)(31).…”