RESUMO
Yeast RNA-binding proteins Nrd1 and Nab3 direct transcription termination of sn/snoRNA transcripts, some mRNA transcripts, and a class of intergenic and anti-sense transcripts. Recognition of Nrd1- and Nab3-binding sites is a critical first step in the termination and subsequent processing or degradation of these transcripts. In this article, we describe the purification and characterization of an Nrd1-Nab3 heterodimer. This Nrd1-Nab3 complex binds specifically to RNA sequences derived from a snoRNA terminator. The relative binding to mutant terminators correlates with the in vivo termination efficiency of these mutations, indicating that the primary specificity determinant in nonpoly(A) termination is Nrd1-Nab3 binding. In addition, several snoRNA terminators contain multiple Nrd1- and Nab3-binding sites and we show that multiple heterodimers bind cooperatively to one of these terminators in vitro.
Assuntos
Proteínas Fúngicas/química , Proteínas de Ligação a RNA/química , RNA/química , Ribonucleoproteínas/química , Regiões Terminadoras Genéticas , Sequência de Bases , Dimerização , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Dados de Sequência Molecular , RNA/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Ribonucleoproteínas/genética , Ribonucleoproteínas/isolamento & purificaçãoRESUMO
The yeast RNA binding proteins Nrd1 and Nab3 are required for termination of nonpolyadenylated transcripts from RNA polymerase (Pol) II-transcribed snRNA and snoRNA genes. In this paper, we show that NRD1 expression is regulated by Nrd1- and Nab3-directed premature termination. Sequences recognized by these proteins are present in NRD1 mRNA and are required for regulated expression. Chromatin immunoprecipitation and transcription run-on experiments show that, in wild-type cells, Pol II occupancy is high at the 5' end of the NRD1 gene and decreases at the 3' end. Mutation of Nrd1 and Nab3 binding sites within the NRD1 mRNA leads to a relative increase in Pol II occupancy of downstream sequences. We further show that NRD1 autoregulation involves components of the exosome and a newly discovered exosome-activating complex. Together, these results show that NRD1 is a eukaryotic cellular gene regulated through premature transcription termination.