Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

ABSTRACT: Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (?247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The ?247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type ?247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of ?247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases ?247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the ?247-Brr2 RP mutants compared with the wild type ?247-Brr2. When stimulated by Prp8, wild type ?247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices.

SUBMITTER: Ledoux S 

PROVIDER: S-EPMC4933248 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature