Accurate and Efficient 3′ Processing of U2 Small Nuclear RNA Precursor in a Fractionated Cytoplasmic Extract

Abstract

The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3′ processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3′ end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3′ end of pre-U2 RNA. The 15S activity did not process the 3′ end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3′ end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA.

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