RNA-binding protein Nrd1 directs poly(A)-independent 3'-end formation of RNA polymerase II transcripts.

A eukaryotic chromosome contains many genes, each transcribed separately by RNA polymerase (pol) I, II or III. Transcription termination between genes prevents the formation of polycistronic RNAs and anti-sense RNAs, which are generally detrimental to the correct expression of genes. Terminating the transcription of protein-coding genes by pol II requires a group of proteins that also direct cleavage and polyadenylation of the messenger RNA in response to a specific sequence element, and are associated with the carboxyl-terminal domain of the largest subunit of pol II (refs 1, 2, 3, 4, 5, 6). By contrast, the cis-acting elements and trans-acting factors that direct termination of non-polyadenylated transcripts made by pol II, including small nucleolar and small nuclear RNAs, are not known. Here we show that read-through transcription from yeast small nucleolar RNA and small nuclear RNA genes into adjacent genes is prevented by a cis-acting element that is recognized, in part, by the essential RNA-binding protein Nrd1. The RNA-binding protein Nab3, the putative RNA helicase Sen1, and the intact C-terminal domain of pol II are also required for efficient response to the element. The same proteins are required for maintaining normal levels of Nrd1 mRNA, indicating that these proteins may control elongation of a subset of mRNA transcripts.

A yeast heterogeneous nuclear ribonucleoprotein complex associated with RNA polymerase II.

Recent evidence suggests a role for the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) in pre-mRNA processing. The yeast NRD1 gene encodes an essential RNA-binding protein that shares homology with mammalian CTD-binding proteins and is thought to regulate mRNA abundance by binding to a specific cis-acting element. The present work demonstrates genetic and physical interactions among Nrd1p, the pol II CTD, Nab3p, and the CTD kinase CTDK-I. Previous studies have shown that Nrd1p associates with the CTD of pol II in yeast two-hybrid assays via its CTD-interaction domain (CID). We show that nrd1 temperature-sensitive alleles are synthetically lethal with truncation of the CTD to 9 or 10 repeats. Nab3p, a yeast hnRNP, is a high-copy suppressor of some nrd1 temperature-sensitive alleles, interacts with Nrd1p in a yeast two-hybrid assay, and coimmunoprecipitates with Nrd1p. Temperature-sensitive alleles of NAB3 are suppressed by deletion of CTK1, a kinase that has been shown to phosphorylate the CTD and increase elongation efficiency in vitro. This set of genetic and physical interactions suggests a role for yeast RNA-binding proteins in transcriptional regulation.

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association.

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3'-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3'-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

Repression of gene expression by an exogenous sequence element acting in concert with a heterogeneous nuclear ribonucleoprotein-like protein, Nrd1, and the putative helicase Sen1.

We have fortuitously identified a nucleotide sequence that decreases expression of a reporter gene in the yeast Saccharomyces cerevisiae 20-fold when inserted into an intron. The primary effect of the insertion is a decrease in pre-mRNA abundance accompanied by the appearance of 3'-truncated transcripts, consistent with premature transcriptional termination and/or pre-mRNA degradation. Point mutations in the cis element relieve the negative effect, demonstrating its sequence specificity. A novel yeast protein, named Nrd1, and a previously identified putative helicase, Sen1, help mediate the negative effect of the cis element. Sen1 is an essential nuclear protein that has been implicated in a variety of nuclear functions. Nrd1 has hallmarks of a heterogeneous nuclear ribonucleoprotein, including an RNA recognition motif, a region rich in RE and RS dipeptides, and a proline- and glutamine-rich domain. An N-terminal domain of Nrd1 may facilitate direct interaction with RNA polymerase II. Disruption of the NRD1 gene is lethal, yet C-terminal truncations that delete the RNA recognition motif and abrogate the negative effect of the cis element nevertheless support cell growth. Thus, expression of a gene containing the cis element could be regulated through modulation of the activity of Nrd1. The recent identification of Nrd1-related proteins in mammalian cells suggests that this potential regulatory pathway is widespread among eukaryotes.

Evidence supporting a tethered tracking model for helicase activity of Escherichia coli Rho factor.

Transcription termination factor Rho of Escherichia coli has an ATP-dependent RNA.DNA helicase activity that presumably facilitates RNA transcript release from the elongation complex. This helicase activity is unidirectional (5' to 3') and is stoichiometric, with one RNA molecule released per Rho hexamer in vitro. A simple RNA tracking model postulates that after Rho's initial binding, it translocates preferentially toward the 3' end of the RNA. Nitrocellulose filter binding studies combined with RNase H cleavage are inconsistent with this simple tracking model. Instead, they support a model in which Rho forms tight primary binding interactions with the recognition region of the RNA and remains bound there while transient secondary RNA binding interactions coupled to ATP hydrolysis serve to scan along the RNA to contact the RNA.DNA helix. This "tethered tracking" model is consistent with other properties of Rho factor, including the presence of two classes of RNA binding sites on the Rho hexamer and the 1:1 stoichiometry in the Rho helicase assay.

A short intervening structure can block rho factor helicase action at a distance.

We have characterized the helicase activity of transcription termination factor rho on a variety of substrates. Helicase activity requires specific recognition of a single-stranded region of RNA upstream (5') of the nucleic acid duplex on which rho acts. Spacer sequences of at least 450 nucleotides can be inserted between the rho-binding signals and the duplex region with little effect on activity. RNA-DNA helices of up to 120 base pairs, but not as long as 210 base pairs, can be disrupted efficiently by rho. The stoichiometry of release of substrates with long spacer sequences, as with the standard substrate, approaches a value of one RNA released per rho hexamer; thus cooperative binding by rho does not account for action at a distance. Instead, these results are consistent with a model in which a single rho hexamer binds initially to terminator sequences and then either loops out or tracks along the intervening RNA to reach the duplex region. Results with complex substrates are inconsistent with looping and support the tracking model: under conditions that allow disruption of RNA-DNA, but not RNA-RNA helices (0.4 mM Mg2+), the presence of a short RNA-RNA helix acts as a block to the disruption of an RNA-DNA helix downstream. These findings are discussed in relation to the mechanism of the helicase activity as well as its role in rho-dependent transcription termination.

Specificity and efficiency of rho-factor helicase activity depends on magnesium concentration and energy coupling to NTP hydrolysis.

The RNA-DNA helicase activity of Escherichia coli transcription termination factor rho can be significantly enhanced at lower potassium chloride and magnesium acetate concentrations than previously used. Decreasing the potassium chloride concentration from 150 to 50 mM increases the rate of release at least 4-fold, while at lower magnesium concentrations less ATP is required for maximal duplex disruption. For all concentrations tested (between 0.1 and 5 mM), the optimal magnesium and ATP concentrations are interdependent; a roughly equimolar ratio gives the maximal rate of RNA release, although peak height and breadth vary. Surprisingly, rho behaves differently with an RNA-RNA duplex, which cannot be efficiently disrupted at magnesium concentrations below 1 mM. Above 2.0 mM, release does occur efficiently suggesting that Mg2+ promotes some structural transition in the RNA-RNA helix to a rho-susceptible conformation. In addition to Mg2+, helicase activity requires hydrolysis of nucleoside triphosphates, but for all four standard NTPs the rates of NTP hydrolysis do not correlate uniformly with the rates of RNA release. Based on the ratio of the rate of RNA release to the rate of NTP hydrolysis, rho utilizes ATP most efficiently. The 2-4-fold weaker coupling of hydrolysis to duplex disruption for the other three NTPs demonstrates that NTP utilization is not, on its own, sufficient for efficient helicase activity. The less efficient coupling with GTP, CTP, and UTP correlates with conformational differences in the protein complex as probed by mild trypsin digestion. The implications of our findings for substrate specificity and energy coupling in the helicase reaction are discussed.