3′ Processing and Termination of Mouse Histone Transcripts Synthesized in vitro by RNA Polymerase II

Xiaohong Gu 0 1 William F. Marzluff 0 1 0 University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA 1 Program in Molecular Biology and Biotechnology, Department of Biochemistry and Biophysics The highly expressed mouse histone H2a-614 gene is located 800 nt 5 of the histone H3-614 gene. There is a 140 nt sequence located 500 nt from the end of the H2-614 mRNA which has been defined as a transcription termination site for RNA polymerase II. We established an in vitro transcription system in which both 3 end processing and transcription termination occur. A template containing the adenovirus major late promoter, a portion of the histone H2a-614 coding region, its 3 processing signal, followed by the transcription termination site was transcribed in a nuclear extract prepared from mouse myeloma cells. Some of the transcripts synthesized in the extract were cleaved at the histone processing site in a reaction which was dependent both on the hairpin binding factor and the U7 snRNP. The efficiency of histone 3 end formation was similar both on synthetic transcripts and transcripts synthesized by RNA polymerase II. Defined transcripts, which were not processed and which mapped to the transcription termination site, were released from the template, suggesting that they were formed by transcription termination. Termination in vitro was dependent on a functional histone processing signal. - The final step in transcription of RNA from the DNA template is termination of transcription and release of both the nascent RNA product and RNA polymerase from the template. Efficient transcription termination is important for recycling RNA polymerase molecules and preventing transcription interference from the upstream run-on transcription. Recent studies have shown that transcription termination is tightly coupled to 3 end processing (reviewed in ref. 1). To understand the molecular basis of transcription termination, it is necessary to have a system in which the various events of RNA metabolism all occur, starting with transcription from a DNA template. As a start in this direction, we have established an in vitro system capable of both 3 end formation and transcription termination from the mouse histone H2a-614 gene. The mechanism of termination by RNA polymerase II (pol II) is not well understood. RNA polymerase II transcribes three * To whom correspondence should be addressed classes of genes: (i) genes encoding the polyadenylated mRNAs, (ii) the replication-dependent histone genes and (iii) the capped small nuclear RNA genes. The 3 end of polyadenylated RNAs is formed by cleavage of the nascent transcript and transcription continues past the cleavage site. Transcription termination on genes encoding polyadenylated mRNAs is dependent on the presence of a functional polyadenylation site (26). In cases where there are two genes which are close together, transcription must terminate between the two genes to prevent disruption of the transcription complex on the downstream promoter by a polymerase transcribing the upstream gene. In several cases where there are two closely positioned genes, transcription termination sites have been identified (7,8). For some genes protein factors which bind the sequence required for transcription termination have been identified (912). In cases where genes are relatively far apart, transcription does not terminate precisely but rather ends in a broad region 3 of the polyadenylation site (13,14). The prevailing model is that a polyadenylation site and a transcription pause site(s) combine to form a complete termination site (15,16). Thus cleavage at the polyadenylation site, followed by degradation of the free 5 end of the nascent RNA by a 53 nuclease, is a critical step in transcription termination (1). Histone mRNAs are the only mRNAs which do not end in a polyA tail (17). The 3 end processing signal contains a 16 nt stemloop followed by a purine-rich U7 snRNP binding site (1821). The 3 end of histone mRNAs is formed by a cleavage reaction between the stemloop and the purine-rich sequence (22), with transcription continuing for at least a few hundred nt past the 3 end of the mRNA (23,24). As in polyadenylated mRNAs, termination of transcription requires a functional histone 3 processing signal (24). The 3 end processing reaction requires a U7 snRNP binding to a purine-rich sequence and a stemloop binding protein (SLBP) which recognizes the stemloop (20,25,26). In one histone gene, the mouse histone H2a-614 gene which is only 800 nt upstream of the H3-614 gene, a transcription termination site has been identified (24). This termination region is 140 nt long, GC-rich (75%), and located 550 nt downstream of the processing site of the H2a-614 gene. For this gene terminated H2a-614 pre-mRNAs were detected in cells suggesting that cleavage of the nascent transcript is not a prerequisite for transcription termination (24). Here, we report conditions that allow both transcription and processing of a transcript from a template that contains both a mouse histone H2a-614 3 processing signal and transcription termination site. In addition to the processed transcripts, we also detected and mapped fulllength transcripts which terminated at the termination site as judged by their release from the supercoiled template. with phenol. The RNA was recovered by ethanol precipitation and analyzed on a 6% polyacrylamide7 M urea gel. The RNA was detected by autoradiography and quantified using a PhosphorImager (Molecular Dynamics). MATERIALS AND METHODS Preparation of nuclear extract Mouse myeloma cells were grown in suspension culture in Dulbeccos Modified Eagles Medium plus 10% horse serum and harvested at a concentration of 46 105 cells/ml. Nuclei were prepared essentially by the method of Shapiro et al. (27), as previously described (28). The nuclei were extracted with varying salt concentrations ranging from 0.22 M KCl, optimal for histone 3 processing, to 0.350.6 M KCl, optimal for in vitro transcription. For low salt extracts, where the nuclei did not break and the chromatin did not swell, the nuclei were removed by centrifugation at 20 000 g for 30 min. For high salt extracts, the chromatin was removed by centrifugation at 100 000 g for 1 h. The resulting supernatant was dialyzed against 20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA and 0.5 mM DTT. Precipitated material was removed by centrifugation, and the supernatant was stored at 80 C in small aliquots. Typical protein concentrations were 46 mg/ml. Transcription in nuclear extracts The HLST gene contains the adenovirus major late promoter (MLP) fused to a portion of the mouse histone H2a-614 gene containing the 3 processing signal, followed by the transcription termination region (Fig. 2A). The genes HLT, HLSTM1 and HLSTM2 were constructed from the HLST gene by substituting the appropriate terminator mutations (HLSTM1 and HLSTM2 genes) or by deleting the 92 nt starting at the Sac (...truncated)