Interaction of yeast RNA-binding proteins Nrd1 and Nab3 with RNA polymerase II terminator elements

Abstract

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.

Yeast RNA-Binding Protein Nab3 Regulates Genes Involved in Nitrogen Metabolism

“…For each of these genes, we observed a peak of Nab3 binding at the 5= end of the transcript. Nab3 forms a heterodimer with Nrd1 (8,16), and many ncRNA terminators require both factors for efficient termination (9,32,41). One of the genes upregulated in response to Nrd1 depletion is the ammonium permease gene MEP2.…”

“…The precise binding sites for Nrd1 and Nab3 can be determined from the presence of T-to-C transitions caused by the 4-thiouracil cross-link (49,50). Using these T-to-C transitions to find the binding sites, we analyzed the top 40 Nab3 binding peaks among NCR genes and found common motifs corresponding to known Nrd1 and Nab3 binding sites (Table S1) (15)(16)(17)(18)(19). Using MEME software (51), we found that the Nrd1 consensus is present in fewer genes but is more statistically significant while the Nab3 consensus is present more often but contains less information (Fig.…”

“…Both coding and noncoding transcripts originate from promoters located in nucleosome-free regions in a process that requires both general transcription factors and gene-specific factors (5-7), but termination of these different classes of Pol II transcripts takes place through two different processes. Stable transcripts like mRNA and SUTs terminate through a process linked to cleavage and polyadenylation while snoRNAs and CUTs terminate through the Nrd1-Nab3-Sen1 (NNS) pathway (8)(9)(10)(11)(12)(13)(14).The NNS complex contains two RNA-binding proteins, Nrd1 and Nab3, which recognize specific sequences in nascent transcripts and direct termination in a process that requires the RNA helicase Sen1 (8,9,(15)(16)(17)(18)(19)(20). NNS interacts with Pol II in part through binding of Nrd1 to phosphorylated Ser5 on the C-terminal domain (CTD) (21-23), a pattern of CTD phosphorylation most prominent in the early stages of the transcription cycle, limiting NNS termination to promoter-proximal transcripts (24-29).…”

“…The NNS complex contains two RNA-binding proteins, Nrd1 and Nab3, which recognize specific sequences in nascent transcripts and direct termination in a process that requires the RNA helicase Sen1 (8,9,(15)(16)(17)(18)(19)(20). NNS interacts with Pol II in part through binding of Nrd1 to phosphorylated Ser5 on the C-terminal domain (CTD) (21)(22)(23), a pattern of CTD phosphorylation most prominent in the early stages of the transcription cycle, limiting NNS termination to promoter-proximal transcripts (24)(25)(26)(27)(28)(29).…”

Transcription termination by nuclear RNA polymerases

“…Importantly, NRD1 and SEN1 mutations cause strong termination defects at some snoRNAs (Kim et al 2006). Nrd1 and Nab3 recognize specific RNA sequences located downstream from the mature 39-end of sn/snoRNAs (GUAA/G and UCUU, respectively) Brow 1996, 1998;Conrad et al 2000;Steinmetz et al 2001;Carroll et al 2004Carroll et al , 2007, and mutations in these sites cause termination defects (Carroll et al 2004). Several snoRNA terminators contain multiple potential binding sites for Ndr1 and Nab3, which form a heterodimer (Carroll et al 2007).…”