Variable effects oftheconserved RNAhairpin element upon3'endprocessing ofhistone pre-mRNA invitro

Wehavestudied therequirements forefficient histonespecific RNA3'processing innuclear extract from mammalian tissue culture cells. Processing isstrongly impaired bymutations inthepre-mRNA spacer element thatreduce thebase-pairing potential withU7RNA. Moreover, byexchanging thehairpin andspacer elements oftwodifferently processed H4genes, we find that this difference isexclusively duetothespacer element. Finally, processing isinhibited bytheaddition ofcompetitor RNAs,ifthese contain awild-type spacer sequence, butnotiftheir spacer element ismutated. Conversely, theimportance ofthehairpin forhistone RNA3'processing ishighly variable: Ahairpin mutant oftheH4-12geneisprocessed withalmost wild-type efficiency inextract fromK21mousemastocytoma cells butisstrongly affected inHeLacellextract, whereas anidentical hairpin mutant oftheH4-1gene isaffected inbothextracts. Thehairpin defect of H4-12-specific RNAinHeLacells canbeovercome by a compensatory mutation thatincreases thebase complementarity toU7snRNA.Verysimilar results werealsoobtained inRNAcompetition experiments: processing ofH4-12-specific RNAcanbecompeted by RNAcarrying awild-type hairpin element inextract fromHeLa,butnotK21cells, whereas processing of H4-1-specific RNAcanbecompeted inbothextracts. Withtwoadditional histone genesweobtained results thatwereinonecaseintermediate andintheother similar tothoseobtained withH4-1.Theseresults suggest that hairpin binding factor(s) cancooperatively support theability ofU7snRNPstoformanactive processing complex, butis(are) notdirectly involved intheprocessing mechanism.