Spacing Constraints on Reinitiation of Paramyxovirus Transcription: The Gene End U Tract Acts as a Spacer to Separate Gene End from Gene Start Sites

Abstract

The paramyxovirus gene end U tracts are thought to serve as templates for the addition of a 3' polyA tail to viral mRNAs. The goal of the work described here was to determine the function in transcription of the naturally occurring variability in length of the gene end U tracts of the paramyxovirus simian virus 5 (SV5). An anchored RT-PCR assay was developed to test the hypothesis that the variable U tracts template the addition of variable lengths of polyA tails to mRNAs. The results showed that although the SV5 NP, M, and SH genes encode U tracts of seven, four, and six U residues, respectively, their mRNAs contain similar polyA tails of approximately 250-290 bases. These results indicate that the variable gene end U tracts are functionally equivalent in directing polyadenylation. A reverse genetics system based on a dicistronic minigenome containing the SH-HN gene junction was used to test the hypothesis that the variable U tracks affect the efficiency of transcription termination. Minigenome templates containing an SH gene end with a long U tract of six residues (U6) directed efficient transcription termination and reinitiation at the downstream HN start site with no nucleotide preference for the downstream intergenic region. Surprisingly, truncating the SH gene end U tract to four residues (U4) did not affect SH termination but, rather, reduced downstream HN reinitiation to 20-30% of wild-type levels. Efficient HN reinitiation could be restored to mutant U4 templates in either of two ways: by increasing the U-tract length from four to six residues or by increasing the length of the intergenic region. Efficient HN reinitiation required a minimum of six bases between the last nucleotide in SH and the first nucleotide in HN. We propose that for some paramyxoviruses, the gene end U tract serves a previously unrecognized role as a spacer region between the gene end and gene start sites.

Cytoplasmic Polyadenylation Element (CPE)- and CPE-binding Protein (CPEB)-independent Mechanisms Regulate Early Class Maternal mRNA Translational Activation in Xenopus Oocytes

“…To address the role of CPEs and CPEB in the control of maternal mRNA cytoplasmic polyadenylation and translational activation, we performed a comparative study of the polyadenylation of multiple endogenous mRNAs from the same sample sets utilizing an RNA ligation-coupled PCR technique (35). Briefly, a DNA oligonucleotide (P1) is ligated directly onto the 3Ј end of all the RNAs in the sample preparation (Fig.…”

“…1A) was a modified version of the technique described previously (35). 4 g of total oocyte RNA, from pools of 5 or 6 oocytes, was ligated to 0.4 g of P1 anchor primer (35), in a 10-l reaction using T4 RNA ligase (New England Biolabs) according to the manufacturer's directions.…”

“…1A) was a modified version of the technique described previously (35). 4 g of total oocyte RNA, from pools of 5 or 6 oocytes, was ligated to 0.4 g of P1 anchor primer (35), in a 10-l reaction using T4 RNA ligase (New England Biolabs) according to the manufacturer's directions. The whole 10-l RNA ligation reaction was used in a 50-l reverse transcription reaction using Superscript III (Invitrogen), according to manufacturer's directions using 0.4 g of P1Ј as the reverse primer (35).…”

“…4 g of total oocyte RNA, from pools of 5 or 6 oocytes, was ligated to 0.4 g of P1 anchor primer (35), in a 10-l reaction using T4 RNA ligase (New England Biolabs) according to the manufacturer's directions. The whole 10-l RNA ligation reaction was used in a 50-l reverse transcription reaction using Superscript III (Invitrogen), according to manufacturer's directions using 0.4 g of P1Ј as the reverse primer (35). 1 l of this cDNA preparation was used in each 50-l PCR using Platinum Taq (Invitrogen), according to the manufacturer's directions.…”

Whole Genome Analysis of Sierra Nevada Virus, a Novel Mononegavirus in the Family Nyamiviridae

“…3,17 A study of the paramyxovirus simian virus 5 (SV5), also a mononegavirus, suggests that the poly-U tract in every termination motif may function as a template for polyadenylation, and a genome spacer separating each gene end from the next gene start. 18 It still remains to be determined whether the same function applies for the transcription termination motifs seen in the Nyamiviridae.…”