2 research outputs found
Human splicing factor SF3a, but not SF1, is essential for pre-mRNA splicing in vivo
The three subunits of human splicing factor SF3a are essential for the formation of the functional 17S U2 snRNP and prespliceosome assembly in vitro. RNAi-mediated depletion indicates that each subunit is essential for viability of human cells. Knockdown of single subunits results in a general block in splicing strongly suggesting that SF3a is a constitutive splicing factor in vivo. In contrast, splicing of several endogenous and reporter pre-mRNAs is not affected after knockdown of SF1, which functions at the onset of spliceosome assembly in vitro and is essential for cell viability. Thus, SF1 may only be required for the splicing of a subset of pre-mRNAs. We also observe a reorganization of U2 snRNP components in SF3a-depleted cells, where U2 snRNA and U2-B'' are significantly reduced in nuclear speckles and the nucleoplasm, but still present in Cajal bodies. Together with the observation that the 17S U2 snRNP cannot be detected in extracts from SF3a-depleted cells, our results provide further evidence for a function of Cajal bodies in U2 snRNP biogenesis
Structure-function analysis of the U2 snRNP-associated splicing factor SF3a
Human splicing factor SF3a is a part of the 17 S U2 snRNP (small nuclear ribonucleoprotein), which interacts with the pre-mRNA branch site early during spliceosome formation. The SF3a subunits of 60, 66 and 120 kDa are all required for SF3a function in vitro. Depletion of individual subunits from HeLa cells by RNA interference results in a global inhibition of splicing, indicating that SF3a is a constitutive splicing factor. Structure-function analyses have defined domains necessary for interactions within the SF3a heterotrimer, association with the U2 snRNP and spliceosome assembly. Studies aimed at the identification of regions in SF3a60 and SF3a66, required for proper intracellular localization, have led to a model for the final steps in U2 snRNP biogenesis and the proposal that SF3a is incorporated into the U2 snRNP in Cajal bodies