Transcription of the human U2 snRNA genes continues beyond the 3′ box in vivo

Abstract

The 3Ј box of the human class II snRNA genes is required for proper 3Ј processing of transcripts, but how it functions is unclear. Several lines of evidence suggest that termination of transcription occurs at the 3Ј box and the terminated transcript is then a substrate for processing. However, using nuclear run-on analysis of endogenous genes, we demonstrate that transcription continues for at least 250 nucleotides beyond the 3Ј box of the U2 genes. Although in vivo footprinting analysis of both the U1 and U2 genes detects no protein-DNA contacts directly over the 3Ј box, a series of G residues immediately downstream from the 3Ј box of the U1 gene are clearly protected from methylation by dimethylsulfate. In conjunction with the 3Ј box of the U1 gene, this in vivo footprinted region causes termination of transcription of transiently transfected U2 constructs, whereas a 3Ј box alone does not. Taken together, these results indicate that the 3Ј box is not an efficient transcriptional terminator but may act as a processing element that is functional in the nascent RNA.

U2 Small Nuclear RNA Is a Substrate for the CCA-adding Enzyme (tRNA Nucleotidyltransferase)

“…Whether the 3Ј box functions as a transcription termination signal or as an RNA processing signal is still unknown (25); however, the first detectable U snRNA precursors have 3Ј-terminal tails that extend nearly to the 3Ј box (22,31,32,35,36,71) and are trimmed by cytoplasmic nucleases that may be related to the yeast multinuclease complex known as the exosome (10,35,36,38).…”

“…Although U2 (but not U1) transcription appears to continue past the 3Ј box (25), it remains to be seen whether the 3Ј box functions as a transcription termination signal, as observed for rRNA transcription by RNA polymerase I (26), or as an RNA processing signal, like the AAUAAA element of polyadenylated mRNAs (27)(28)(29) and the CAGA box of nonpolyadenylated replicative histone mRNAs (30). U2ϩ10 is subsequently exported to the cytoplasm, where the 3Ј tail is trimmed (31)(32)(33)(34) by endonuclease and exonuclease activities that could be related to the yeast exosome, a multinuclease complex involved in processing many cellular RNAs (35,36).…”

Transcription termination by nuclear RNA polymerases

“…Formation of their 39-ends depends on specific snRNA promoters but does not require a poly(A) site or HDE, like histone mRNAs, but rather a 39-box element located 9-19 nt downstream from the mature 39-end of snRNAs (de Vegvar et al 1986;Hernandez and Weiner 1986). The 39-box is important for proper transcription of snRNAs and was shown to be required for 39-end processing of U1 and U2 snRNAs (Cuello et al 1999). After endonucleolytic cleavage downstream from the 39-box, snRNAs carry an extended 39-end that is trimmed after their export to the cytoplasm (for review, see ).…”

“…While transcription of U1 genes terminates close to the 39-box (Cuello et al 1999), U2 transcription terminates ;1 kb from its mature 39-end (Medlin et al 2003). An unidentified termination factor or complex appears to bind the U1 precursor RNA immediately downstream from the 39-box at a G track, just upstream of a T stretch (Cuello et al 1999). When this potential terminator-binding region is positioned downstream from the U2 39-box, it leads to efficient transcription termination.…”

The linked units of 5S rDNA and U1 snDNA of razor shells (Mollusca: Bivalvia: Pharidae)

“…Linked units of 5S rDNA and U1 snDNA J Vierna et al human U1 snDNA terminates close to the 3 0 box (Cuello et al, 1999), but transcription of U2 snDNA extends about 800 sites beyond it (Medlin et al, 2003). The human 3 0 box (Hernandez, 1985) is a 16 nt stretch, located 10 sites downstream the U1.…”