Multiple transcripts from a 3'-UTR reporter vary in sensitivity to nonsense-mediated mRNA decay in Saccharomyces cerevisiae.

Nonsense-mediated mRNA decay (NMD) causes accelerated transcript degradation when a premature translation termination codon disrupts the open reading frame (ORF). Although endogenous transcripts that have uninterrupted ORFs are typically insensitive to NMD, some can nonetheless become prone to NMD when translation terminates at out-of-frame premature stop codons. This occurs when introns containing stop codons fail to be spliced, when translation of an upstream ORF (uORF) terminates in the 5'-untranslated region (5'-UTR) or the coding region, or when the 5'-proximal AUG initiation codon is bypassed and translation initiates at a downstream out-of-frame AUG followed by a stop codon. Some 3'-untranslated regions (3'-UTRs) are also known to trigger NMD, but the mechanism is less well understood. To further study the role of 3'-UTRs in NMD, a reporter system was designed to examine 3'-UTRs from candidate genes known to produce NMD-sensitive transcripts. Out of eight that were tested, the 3'-UTRs from MSH4 and SPO16 caused NMD-dependent mRNA destabilization. Both endogenous genes produce multiple transcripts that differ in length at the 3' end. Detailed studies revealed that the longest of six reporter MSH4-3'-UTR transcripts was NMD-sensitive but five shorter transcripts were insensitive. NMD-dependent degradation of the long transcript required Xrn1, which degrades mRNA from the 5' end. Sensitivity to NMD was not associated with extensive translational read-through past the normal stop codon. To our knowledge, this is the first example where multiple transcripts containing the same ORF are differentially sensitive to NMD in Saccharomyces cerevisiae. The results provide a proof of principle that long 3'-UTRs can trigger NMD, which suggests a potential link between errors in transcription termination or processing and mRNA decay.

Interactions of Sen1, Nrd1, and Nab3 with multiple phosphorylated forms of the Rpb1 C-terminal domain in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SEN1 gene codes for a nuclear, ATP-dependent helicase which is embedded in a complex network of protein-protein interactions. Pleiotropic phenotypes of mutations in SEN1 suggest that Sen1 functions in many nuclear processes, including transcription termination, DNA repair, and RNA processing. Sen1, along with termination factors Nrd1 and Nab3, is required for the termination of noncoding RNA transcripts, but Sen1 is associated during transcription with coding and noncoding genes. Sen1 and Nrd1 both interact directly with Nab3, as well as with the C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II. It has been proposed that Sen1, Nab3, and Nrd1 form a complex that associates with Rpb1 through an interaction between Nrd1 and the Ser5-phosphorylated (Ser5-P) CTD. To further study the relationship between the termination factors and Rpb1, we used two-hybrid analysis and immunoprecipitation to characterize sen1-R302W, a mutation that impairs an interaction between Sen1 and the Ser2-phosphorylated CTD. Chromatin immunoprecipitation indicates that the impairment of the interaction between Sen1 and Ser2-P causes the reduced occupancy of mutant Sen1 across the entire length of noncoding genes. For protein-coding genes, mutant Sen1 occupancy is reduced early and late in transcription but is similar to that of the wild type across most of the coding region. The combined data suggest a handoff model in which proteins differentially transfer from the Ser5- to the Ser2-phosphorylated CTD to promote the termination of noncoding transcripts or other cotranscriptional events for protein-coding genes.

Cyclin-dependent kinase-like function is shared by the beta- and gamma- subset of the conserved herpesvirus protein kinases.

The UL97 protein of human cytomegalovirus (HCMV, or HHV-5 (human herpesvirus 5)), is a kinase that phosphorylates the cellular retinoblastoma (Rb) tumor suppressor and lamin A/C proteins that are also substrates of cellular cyclin-dependent kinases (Cdks). A functional complementation assay has further shown that UL97 has authentic Cdk-like activity. The other seven human herpesviruses each encode a kinase with sequence and positional homology to UL97. These UL97-homologous proteins have been termed the conserved herpesvirus protein kinases (CHPKs) to distinguish them from other human herpesvirus-encoded kinases. To determine if the Cdk-like activities of UL97 were shared by all of the CHPKs, we individually expressed epitope-tagged alleles of each protein in human Saos-2 cells to test for Rb phosphorylation, human U-2 OS cells to monitor nuclear lamina disruption and lamin A phosphorylation, or S. cerevisiae cdc28-13 mutant cells to directly assay for Cdk function. We found that the ability to phosphorylate Rb and lamin A, and to disrupt the nuclear lamina, was shared by all CHPKs from the beta- and gamma-herpesvirus families, but not by their alpha-herpesvirus homologs. Similarly, all but one of the beta and gamma CHPKs displayed bona fide Cdk activity in S. cerevisiae, while the alpha proteins did not. Thus, we have identified novel virally-encoded Cdk-like kinases, a nomenclature we abbreviate as v-Cdks. Interestingly, we found that other, non-Cdk-related activities reported for UL97 (dispersion of promyelocytic leukemia protein nuclear bodies (PML-NBs) and disruption of cytoplasmic or nuclear aggresomes) showed weak conservation among the CHPKs that, in general, did not segregate to specific viral families. Therefore, the genomic and evolutionary conservation of these kinases has not been fully maintained at the functional level. Our data indicate that these related kinases, some of which are targets of approved or developmental antiviral drugs, are likely to serve both overlapping and non-overlapping functions during viral infections.

Sen1p performs two genetically separable functions in transcription and processing of U5 small nuclear RNA in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SEN1 gene codes for a nuclear-localized superfamily I helicase. SEN1 is an ortholog of human SETX (senataxin), which has been implicated in the neurological disorders ataxia-ocular apraxia type 2 and juvenile amyotrophic lateral sclerosis. Pleiotropic phenotypes conferred by sen1 mutations suggest that Sen1p affects multiple steps in gene expression. Sen1p is embedded in a protein-protein interaction network involving direct binding to multiple partners. To test whether the interactions occur independently or in a dependent sequence, we examined interactions with the RNA polymerase II subunit Rpb1p, which is required for transcription, and Rnt1p, which is required for 3'-end maturation of many noncoding RNAs. Mutations were identified that impair one of the two interactions without impairing the other interaction. The effects of the mutants on the synthesis of U5 small nuclear RNA were analyzed. Two defects were observed, one in transcription termination and one in 3'-end maturation. Impairment of the Sen1p-Rpb1p interaction resulted in a termination defect. Impairment of the Sen1p-Rnt1p interaction resulted in a processing defect. The results suggest that the Sen1p-Rpb1p and Sen1p-Rnt1p interactions occur independently of each other and serve genetically separable purposes in targeting Sen1p to function in two temporally overlapping steps in gene expression.

Nonsense-mediated decay of ash1 nonsense transcripts in Saccharomyces cerevisiae.

Nonsense-mediated mRNA decay (NMD) performs two functions in eukaryotes, one in controlling the expression level of a substantial subset of genes and the other in RNA surveillance. In the vast majority of genes, nonsense mutations render the corresponding transcripts prone to surveillance and subject to rapid degradation by NMD. To examine whether some classes of nonsense transcripts escape surveillance, we asked whether NMD acts on mRNAs that undergo subcellular localization prior to translation. In Saccharomyces cerevisiae, wild-type ASH1 mRNA is one of several dozen transcripts that are exported from the mother-cell nucleus during mitotic anaphase, transported to the bud tip on actin cables, anchored at the bud tip, and translated. Although repressed during transport, translation is a prerequisite for NMD. We found that ash1 nonsense mutations affect transport and/or anchoring independently of NMD. The nonsense transcripts respond to NMD in a manner dependent on the position of the mutation. Maximal sensitivity to NMD occurs when transport and translational repression are simultaneously impaired. Overall, our results suggest a model in which ash1 mRNAs are insensitive to NMD while translation is repressed during transport but become sensitive once repression is relieved.

Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function.

As obligate intracellular parasites, viruses expertly modify cellular processes to facilitate their replication and spread, often by encoding genes that mimic the functions of cellular proteins while lacking regulatory features that modify their activity. We show that the human cytomegalovirus UL97 protein has activities similar to cellular cyclin-cyclin-dependent kinase (CDK) complexes. UL97 phosphorylated and inactivated the retinoblastoma tumor suppressor, stimulated cell cycle progression in mammalian cells, and rescued proliferation of Saccharomyces cerevisiae lacking CDK activity. UL97 is not inhibited by the CDK inhibitor p21 and lacks amino acid residues conserved in the CDKs that permit the attenuation of kinase activity. Thus, UL97 represents a functional ortholog of cellular CDKs that is immune from normal CDK control mechanisms.

Detecting phosphorylation-dependent interactions with the C-terminal domain of RNA polymerase II subunit Rpb1p using a yeast two-hybrid assay.

Rpb1p, the largest subunit of S. cerevisiae RNA polymerase II, contains a repetitive structure called the C-terminal domain (CTD). The CTD serves as a scaffold for the regulated association and dissociation of more than a hundred proteins involved in RNA synthesis. Phosphorylation of two serine residues (Ser(2) and Ser(5)) in the repeating units of the CTD change dynamically during the pre-initiation, initiation, elongation and termination of transcription to control the binding and release of transcriptional components. A modification of the well established yeast two-hybrid assay for protein-protein interactions is described that detects interactions between phosphorylated forms of the CTD and proteins whose interactions with the CTD depend on phosphorylation. The efficacy of the approach was established by first showing that two-hybrid fusions containing the CTD are phosphorylated at Ser(2) and Ser(5) residues. Interactions between the CTD and three known CTD-binding proteins were analyzed. The results suggest that the modified two-hybrid system accurately assays CTD-binding and provides a new and convenient assay for CTD-binding proteins.

Impact of nonsense-mediated mRNA decay on the global expression profile of budding yeast.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd(+)) and mutant (Nmd(-)) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% +/- 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd(-) strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are available.

Ebs1p, a negative regulator of gene expression controlled by the Upf proteins in the yeast Saccharomyces cerevisiae.

Mutations in EBS1 were identified in Saccharomyces cerevisiae that cosuppress missense, frameshift, and nonsense mutations. Evidence from studies of loss of function and overexpression of EBS1 suggests that Ebs1p affects gene expression by inhibiting translation and that a loss of EBS1 function causes suppression by increasing the rate of translation. Changes in EBS1 expression levels alter the expression of wild-type genes, but, in general, no changes in mRNA abundance were associated with a loss of function or overexpression of EBS1. Translation of a lacZ reporter was increased in strains carrying an ebs1-Delta mutant gene, whereas translation was decreased when EBS1 was overexpressed. The cap binding protein eIF-4E copurifies with Ebs1p in the absence of RNA, suggesting that the two proteins interact in vivo. Although physical and genetic interactions were detected between Ebs1p and Dcp1p, copurification was RNase sensitive, and changes in the expression of Ebs1p had little to no effect on decapping of the MFA2 transcript. The combined results suggest that Ebs1p inhibits translation, most likely through effects on eIF-4E rather than on decapping. Finally, EBS1 transcript levels are under the control of nonsense-mediated mRNA decay (NMD), providing the first example of an NMD-sensitive transcript whose protein product influences a step in gene expression required for NMD.

Transcript selection and the recruitment of mRNA decay factors for NMD in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, nonsense-mediated mRNA decay (NMD) requires Upf1p, Upf2p, and Upf3p to accelerate the decay rate of two unique classes of transcripts: (1) nonsense mRNAs that arise through errors in gene expression, and (2) naturally occurring transcripts that lack coding errors but have built-in features that target them for accelerated decay (error-free mRNAs). NMD can trigger decay during any round of translation and can target Cbc-bound or eIF-4E-bound transcripts. Extremely low concentrations of the Upf proteins relative to the total pool of transcripts make it difficult to understand how nonsense transcripts are selectively recruited. To stimulate debate, we propose two alternative mechanisms for selecting nonsense transcripts for NMD and for assembling components of the surveillance complex, one for the first (pioneer) round of translation, called "nuclear marking," and the other for subsequent rounds, called "reverse assembly." The model is designed to accommodate (1) the low abundance of NMD factors, (2) the role of nucleocytoplasmic shuttling proteins in NMD, (3) the independent and nonobligate order of assembly of two different subcomplexes of NMD factors, and (4) the ability of NMD to simultaneously reduce or eliminate the synthesis of truncated proteins produced by nonsense transcripts while down-regulating but not completely eliminating functional proteins produced from error-free NMD-sensitive transcripts

Using the cre-lox recombination system to assess functional impairment caused by amino acid substitutions in yeast proteins.

A method was developed to assess the functional significance of a sequence motif in yeast Upf3p, a protein required for nonsense-mediated mRNA decay (NMD). The motif lies at the edge of the Upf3p-Upf2p interaction domain, but at the same time resembles the canonical leucine-rich nuclear export sequence (NES) found in proteins that bind Crm1p exportin. To test the function of the putative NES, site-directed mutations that cause substitutions of conserved NES-A residues were first selected to identify hypermorphic alleles. Next, a portable Crm1p-binding NES from HIV-1 Rev protein that functions in yeast was fused en masse to the C-terminus of variant Upf3 proteins using loxP sites recognized by bacterial cre-recombinase. Finally, variant Upf3-Rev proteins that were functional in NMD were selected and examined for the types of amino acid substitutions present in NES-A. The mutational analysis revealed that amino acid substitutions in the Upf3 NES impair both nuclear export and the Upf2p-Upf3p interaction, both of which are required for Upf3p to function in NMD. The method described in this report could be modified for the genetic analysis of a variety of portable protein domains.

Multiple protein/protein and protein/RNA interactions suggest roles for yeast DNA/RNA helicase Sen1p in transcription, transcription-coupled DNA repair and RNA processing.

Sen1p in Saccharomyces cerevisiae is a Type I DNA/RNA helicase. Mutations in the helicase domain perturb accumulation of diverse RNA classes, and Sen1p has been implicated in 3' end formation of non-coding RNAs. Using a combination of global and candidate-specific two hybrid screens, eight proteins were identified that interact with Sen1p. Interactions with three of the proteins were analyzed further: Rpo21p(Rpb1p), a subunit of RNA polymerase II, Rad2p, a deoxyribonuclease required in DNA repair, and Rnt1p (RNase III), an endoribonuclease required for RNA maturation. For all three interactions, the two-hybrid results were confirmed by co-immunoprecipitation experiments. Genetic tests designed to assess the biological significance of the interactions indicate that Sen1p plays functionally significant roles in transcription and transcription-coupled DNA repair. To investigate the potential role of Sen1p in RNA processing and to assess the functional significance of the Sen1p/Rnt1p interaction, we examined U5 snRNA biogenesis. We provide evidence that Sen1p functions in concert with Rnt1p and the exosome at a late step in 3' end formation of one of the two mature forms of U5 snRNA but not the other. The protein-protein and protein-RNA interactions reported here suggest that the DNA/RNA helicase activity of Sen1p is utilized for several different purposes in multiple gene expression pathways.

Looking at mRNA decay pathways through the window of molecular evolution.

In eukaryotes, mRNAs are monitored for errors in gene expression by RNA surveillance where untranslatable mRNAs are selectively degraded by the nonsense-mediated mRNA decay (NMD) pathway. Depending on the organism, three to seven genes are required for NMD. Besides RNA surveillance, the genes required for NMD serve a second purpose by controlling the overall abundance of a substantial fraction of the transcriptome.

mRNAs encoding telomerase components and regulators are controlled by UPF genes in Saccharomyces cerevisiae.

Telomeres, the chromosome ends, are maintained by a balance of activities that erode and replace the terminal DNA sequences. Furthermore, telomere-proximal genes are often silenced in an epigenetic manner. In Saccharomyces cerevisiae, average telomere length and telomeric silencing are reduced by loss of function of UPF genes required in the nonsense-mediated mRNA decay (NMD) pathway. Because NMD controls the mRNA levels of several hundred wild-type genes, we tested the hypothesis that NMD affects the expression of genes important for telomere functions. In upf mutants, high-density oligonucleotide microarrays and Northern blots revealed that the levels of mRNAs were increased for genes encoding the telomerase catalytic subunit (Est2p), in vivo regulators of telomerase (Est1p, Est3p, Stn1p, and Ten1p), and proteins that affect telomeric chromatin structure (Sas2p and Orc5p). We investigated whether overexpressing these genes could mimic the telomere length and telomeric silencing phenotypes seen previously in upf mutant strains. Increased dosage of STN1, especially in combination with increased dosage of TEN1, resulted in reduced telomere length that was indistinguishable from that in upf mutants. Increased levels of STN1 together with EST2 resulted in reduced telomeric silencing like that of upf mutants. The half-life of STN1 mRNA was not altered in upf mutant strains, suggesting that an NMD-controlled transcription factor regulates the levels of STN1 mRNA. Together, these results suggest that NMD maintains the balance of gene products that control telomere length and telomeric silencing primarily by maintaining appropriate levels of STN1, TEN1, and EST2 mRNA.

Nuclear import of Upf3p is mediated by importin-alpha/-beta and export to the cytoplasm is required for a functional nonsense-mediated mRNA decay pathway in yeast.

Upf3p, which is required for nonsense-mediated mRNA decay (NMD) in yeast, is primarily cytoplasmic but accumulates inside the nucleus when UPF3 is overexpressed or when upf3 mutations prevent nuclear export. Upf3p physically interacts with Srp1p (importin-alpha). Upf3p fails to be imported into the nucleus in a temperature-sensitive srp1-31 strain, indicating that nuclear import is mediated by the importin-alpha/beta heterodimer. Nuclear export of Upf3p is mediated by a leucine-rich nuclear export sequence (NES-A), but export is not dependent on the Crm1p exportin. Mutations identified in NES-A prevent nuclear export and confer an Nmd(-) phenotype. The addition of a functional NES element to an export-defective upf(-) allele restores export and partially restores an Nmd(+) phenotype. Our findings support a model in which the movement of Upf3p between the nucleus and the cytoplasm is required for a fully functional NMD pathway. We also found that overexpression of Upf2p suppresses the Nmd(-) phenotype in mutant strains carrying nes-A alleles but has no effect on the localization of Upf3p. To explain these results, we suggest that the mutations in NES-A that impair nuclear export cause additional defects in the function of Upf3p that are not rectified by restoration of export alone.