RESUMEN
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.
Asunto(s)
Genes Virales/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Respirovirus/genética , Transcripción Genética/genética , Animales , Secuencia de Bases , Línea Celular , Perros , Variación Genética/genética , Genoma Viral , Modelos Genéticos , Poli A/genética , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Moldes Genéticos , Regiones Terminadoras Genéticas/genéticaRESUMEN
AUF1 is a RNA-binding protein that contains two non-identical RNA recognition motifs (RRMs). AUF1 binds to A + U-rich elements (AREs) with high affinity. The binding of AUF1 to AREs is believed to serve as a signal to a mRNA processing pathway which degrades mRNAs encoding many cytokines, oncoproteins and G protein-coupled receptors. Because the ARE-binding activity of AUF1 appears central to the regulation of many important genes, we analyzed the domains of the protein that are important for this activity. Examination of the binding affinity of various mutants indicates that both RRMs may be required for binding. However, they are not sufficient. Highest affinity binding requires an alanine/glycine-rich region of the N-terminus and a short glutamine-rich region in the C-terminus. The N-terminus is required for dimerization of AUF1. However, AUF1 binds an ARE as a hexameric protein. Thus protein-protein interactions are important for high affinity ARE-binding activity of AUF1.