ATP-dependent remodeling of the spliceosome: intragenic suppressors of release-defective mutants of Saccharomyces cerevisiae Prp22.
The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (635SAT637) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (635AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (804QAKGRAGR811). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A.
Campodonico
Eva
E
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA.
Schwer
Beate
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D053487:DEAD-box RNA Helicases; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D020365:RNA Helicases; D000072260:RNA Splicing Factors; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes / Q000502:physiology*; D013696:Temperature
D005656:Fungal Proteins; D000072260:RNA Splicing Factors; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C067135:PRP22 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases