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.

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.