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 H (Pol H)-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 plwmbaginifolia. We found that the 3'-end-adjacent sequence CA (N)310AGTNNAA, conserved in plant Pol lI-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 lI-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 r-specific plant U snRNA gene promoter is not compatible with polyadenylation. Finally, we reveal that initiation at a Pol H-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.

An upstream U-snRNA gene-like promoter is required for transcription of theArabidopsis thaliana7SL RNA gene

“…The same mutation was previously shown to abolish Arabidopsis U6 gene transcription in vivo (26,27). While the point mutations made in the USE sequence of At7SL-2 were not previously tested in pol Ill-specific U-snRNA genes, it has been observed that a different point mutation in the USE had similar effects on both pol II and pol III U-snRNA transcription in plant protoplasts (33). In agreement with this finding, the 20-fold decrease in At7SL-2 gene expression resulting from the USE-Dn mutation is similar to that reported for the pol 11-specific Arabidopsis U2 snRNA gene (25).…”

Pac1p, an RNase III homolog, is required for formation of the 3′ end of U2 snRNA in Schizosaccharomyces pombe

“…We have shown that Pac1p is required for the processing of rRNA precursors in S. pombe cells and Rotondo et al+ (1997) have demonstrated that the 39 ETS is cleaved by this enzyme in vitro at known in vivo processing sites (Melekhovets et al+, 1994)+ As was shown for the extended U2 transcripts in the S. cerevisiae rnt 1 mutant (Abou Elela & Ares, 1998), the long U2 transcripts seen in ts138 at the restrictive temperature are polyadenylated (data not shown)+ Although an RNase III-like protein, Rnt1p, is also required for formation of snRNA 39 ends and cleavage of the 39 ETS of rRNA precursors in S. cerevisiae, there are some differences in snRNA and rRNA synthesis between the two yeasts+ In the S. cerevisiae rnt1 mutant, the accumulation of U2 and U5L RNAs is reduced at the normal and restrictive temperatures, whereas the processing of rRNA precursors is deficient only at the restrictive temperature (Abou Elela et al+, 1996; Chanfreau et al+, 1997)+ In contrast, in the pac1-A342T mutants described here, the defects in snRNA and rRNA synthesis are manifest only at the restrictive temperature+ Thus the relative affinities of snRNA and rRNA precursors for each RNase III homolog may be different, or the differences between the yeasts may reflect the nature of the mutations within their respective RNase III homologs+ The sequence of the rnt1 mutation has not been published+ In rnt1 cells the steady-state levels of U1, U4, and U5S snRNAs is increased at the restrictive temperature (Chanfreau et al+, 1997), whereas in the pac1-A342T mutants, the synthesis of these snRNAs is decreased (Fig+ 4)+ Thus, in S. cerevisiae there appears to be an alternative pathway for formation of the 39 ends of RNA polymerase II-transcribed snRNAs+ Such a pathway either does not exist or is very minor in S. pombe, and 39-end formation of the major snRNAs in this organism appears to require Pac1p+ In addition, at least one other component is involved in snRNA synthesis in S. pombe, namely the factor mutated in ts35+ As purified Pac1p, alone, can cleave U2 precursors in vitro, the role of the factor mutated in ts35 is not clear and awaits the cloning of the corresponding gene+ Our results show that in S. pombe, as in S. cerevisiae (Chanfreau et al+, 1997;Abou Elela & Ares, 1998), the formation of the 39 ends of snRNAs can be dissociated from transcription+ Studies in plants (Connelly & Filipowicz, 1993) and sea urchins (Wendelburg & Marzluff, 1992) indicate that the two processes may also be separable in these organisms+ In contrast, in vertebrates 39-end formation is intimately coupled to tran-scription from snRNA promoters (Hernandez & Weiner, 1986;Neuman de Vegvar et al+, 1986)+ However, there are some similarities between the yeast and vertebrate systems+ In S. cerevisiae (Noble & Guthrie, 1996) and in extracts from S. pombe cells (Fig+ 7), cleavage of snRNA precursors by an RNase III homolog gives rise to intermediates with short extensions that are subsequently trimmed away+ Such intermediates in snRNA metabolism were first observed in mammalian cells …”

Small nuclear RNA genes transcribed by either RNA polymerase II or RNA polymerase III in monocot plants share three promoter elements and use a strategy to regulate gene expression different from that used by their dicot plant counterparts.

“…This contrasts with the situation in dicot plant protoplasts, in which activity of the promoter is a function of the quality of the USE element. In dicot plant genes, the USE sequence (consensus TCCCACATCG) is very highly conserved, and as expected, point mutations in the element usually decrease gene transcription by as much as 75 to 95% (5,13,38). On the other hand, USE elements in all monocot snRNA genes deviate in at least one and often in two or three positions from the TCCCACATCG sequence (Fig.…”

“…Plant genes contain two promoter elements, the snRNA GENES TRANSCRIBED BY RNA Pol II OR RNA Pol III 5911 upstream sequence element (USE; consensus TCCCACA TCG) and a -30 TATA box (reviewed in references 13 and 33). Detailed analysis of transcription of U2 and U6 genes of A. thaliana has revealed that these two elements are necessary and sufficient for transcription initiation in transfected protoplasts (5,13,38,40). The USE is a highly conserved plant snRNA gene-specific element, and its deletion or mutation decreases transcription efficiency 10-to 20-fold (38,40).…”