title: Activity of chimeric U small nuclear RNA (snRNA)/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U snRNA 3'-end formation and transcription initiation can occur independently in plants.
abstract: Formation of the 3' ends of RNA polymerase II (Pol II)-specific U small nuclear RNAs (U snRNAs) in vertebrate cells is dependent upon transcription initiation from the U snRNA gene promoter. Moreover, U snRNA promoters are unable to direct the synthesis of functional polyadenylated mRNAs. In this work, we have investigated whether U snRNA 3'-end formation and transcription initiation are also coupled in plants. We have first characterized the requirements for 3'-end formation of an Arabidopsis U2 snRNA expressed in transfected protoplasts of Nicotiana plumbaginifolia. We found that the 3'-end-adjacent sequence CA (N)3-10AGTNNAA, conserved in plant Pol II-specific U snRNA genes, is essential for the 3'-end formation of U2 transcripts and, similar to the vertebrate 3' box, is highly tolerant to mutation. The 3'-flanking regions of an Arabidopsis U5 and a maize U2 snRNA gene can effectively substitute for the Arabidopsis U2 3'-end formation signal, indicating that these signals are functionally equivalent among different Pol II-transcribed snRNA genes. The plant U snRNA 3'-end formation signal can be recognized irrespective of whether transcription initiation occurs at U snRNA or mRNA gene promoters, although efficiency of 3' box utilization is higher when transcription initiation occurs at the U snRNA promoter. Moreover, transcripts initiated from the U2 gene promoter can be spliced and polyadenylated. Transcription from a Pol III-specific plant U snRNA gene promoter is not compatible with polyadenylation. Finally, we reveal that initiation at a Pol II-specific plant U snRNA gene promoter can occur in the absence of the snRNA coding region and a functional snRNA 3'-end formation signal, demonstrating that these sequences play no role in determining the RNA polymerase specificity of plant U snRNA genes.
authors.affiliation: Friedrich Miescher Institut, Basel, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Connelly
authors.forename: W
authors.initials: W
authors.lastname: Filipowicz
mesh_terms: D017360:Arabidopsis; D001483:Base Sequence; D002678:Chimera; D003001:Cloning, Molecular; D004247:DNA; D017343:Genes, Plant; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D011061:Poly A; D011401:Promoter Regions, Genetic; D011523:Protoplasts / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D011061:Poly A; D004247:DNA; D012319:RNA Polymerase II