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Vol. 16, Issue 3, 1366-1377, March 2005

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Human Splicing Factor SF3a, but Not SF1, Is Essential for Pre-mRNA Splicing In Vivo

Department of Cell Biology, Faculty of Sciences, University of Geneva, CH-1211 Geneva 4, Switzerland

Submitted November 30, 2004; Revised December 28, 2004; Accepted December 29, 2004
Monitoring Editor: Joseph Gall

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.

Abbreviations used: AMCA, 7-amino-4-methylcoumarin-3-acetic acid; BrdU, bromo-desoxy-uridine; CB, Cajal body; FISH, fluorescence in situ hybridization; FU, fluoro-uridine; RNAi, RNA interference; RNA pol II, RNA polymerase II; siRNA, small interfering RNA; SMN, survival of motor neurons; snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein particle; WT1, Wilms' tumor 1.

Address correspondence to: Angela Krämer (angela.kraemer{at}cellbio.unige.ch).

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