mRNAs are monitored for errors in gene expression by RNA surveillance, in which mRNAs that cannot be fully translated are degraded by the nonsense-mediated mRNA decay pathway (NMD). RNA surveillance ensures that potentially deleterious truncated proteins are seldom made. NMD pathways that promote surveillance have been found in a wide range of eukaryotes. In Saccharomyces cerevisiae, the proteins encoded by the UPF1, UPF2, and UPF3 genes catalyze steps in NMD and are required for RNA surveillance. In this report, we show that the Upf proteins are also required to control the total accumulation of a large number of mRNAs in addition to their role in RNA surveillance. High-density oligonucleotide arrays were used to monitor global changes in the yeast transcriptome caused by loss of UPF gene function. Null mutations in the UPF genes caused altered accumulation of hundreds of mRNAs. The majority were increased in abundance, but some were decreased. The same mRNAs were affected regardless of which of the three UPF gene was inactivated. The proteins encoded by UPF-dependent mRNAs were broadly distributed by function but were underrepresented in two MIPS (Munich Information Center for Protein Sequences) categories: protein synthesis and protein destination. In a UPF(+) strain, the average level of expression of UPF-dependent mRNAs was threefold lower than the average level of expression of all mRNAs in the transcriptome, suggesting that highly abundant mRNAs were underrepresented. We suggest a model for how the abundance of hundreds of mRNAs might be controlled by the Upf proteins.

In yeast, the accelerated rate of decay of nonsense mutant mRNAs, called nonsense-mediated mRNA decay, requires three proteins, Upf1p, Upf2p, and Upf3p. Single, double, and triple disruptions of the UPF genes had nearly identical effects on nonsense mRNA accumulation, suggesting that the encoded proteins function in a common pathway. We examined the distribution of epitope-tagged versions of Upf proteins by sucrose density gradient fractionation of soluble lysates and found that all three proteins co-distributed with 80 S ribosomal particles and polyribosomes. Treatment of lysates with RNase A caused a coincident collapse of polyribosomes and each Upf protein into fractions containing 80 S ribosomal particles, as expected for proteins that are associated with polyribosomes. Mutations in the cysteine-rich (zinc finger) and RNA helicase domains of Upf1p caused loss of function, but the mutant proteins remained polyribosome-associated. Density gradient profiles for Upf1p were unchanged in the absence of Upf3p, and although similar, were modestly shifted to fractions lighter than those containing polyribosomes in the absence of Upf2p. Upf2p shifted toward heavier polyribosome fractions in the absence of Upf1p and into fractions containing 80 S particles and lighter fractions in the absence of Upf3p. Our results suggest that the association of Upf2p with polyribosomes typically found in a wild-type strain depends on the presence and opposing effects of Upf1p and Upf3p.The notion of a global pathway for eukaryotic mRNA decay suggested by early work in animal cells has recently been greatly advanced by studies in the yeast Saccharomyces cerevisiae (1-4). Using an in vivo transcriptional pulse, the temporal fate of newly synthesized mRNA was established by monitoring poly(A) tail length, loss of the m 7 Gppp cap, disappearance of the mRNA, and the appearance of degradation intermediates. mRNAs with shorter half-lives were generally subject to faster rates of deadenylation and decapping. Once the poly(A) tails were reduced to a short oligo(A) length (10 -12 nucleotides), the mRNAs were decapped and digested from the 5Ј end. Decapping requires Dcp1p (5). Processive degradation from the 5Ј end requires the product of the XRN1 gene, which is known to encode a 5Ј 3 3Ј exoribonuclease (6). Deadenylation-dependent decapping followed by 5Ј 3 3Ј exonucleolytic decay is likely to be the global default pathway for the degradation of most eukaryotic mRNAs.Yeast mRNAs containing a premature stop codon decay more rapidly than their wild-type counterparts (7). This accelerated decay, called nonsense-mediated mRNA decay (NMD), 1 requires cis-acting elements in the mRNA in addition to a premature stop codon (8). Premature translational termination triggers decapping at the 5Ј end of nonsense mRNAs with kinetics that are independent of deadenylation (9). Following decapping, decay proceeds through the Xrn1p-mediated nucleolysis that is common to intrinsic decay. These results support the view that when translation is prematurely terminat...

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.Telomeres, the ends of linear chromosomes, are important for chromosome integrity and are maintained by telomerase, a reverse transcriptase-like enzyme that includes an integral RNA template. The catalytic components of Saccharomyces cerevisiae telomerase (TLC1 RNA and Est2p), as well as gene products required for telomerase activity in vivo (e.g., Est1p, Est3p, Cdc13/Est4p, Ku70/80, Mec1p, MRX, Rap1p, Stn1p, Tel1p, and Ten1p), have been identified (reviewed in reference 12). However, mechanisms that regulate the expression and activity of telomerase components and modulators have not been explored.The nonsense-mediated mRNA decay (NMD) pathway accelerates the degradation of mRNAs that prematurely terminate translation due to nonsense mutations, frameshifts, or translation of alternate open reading frames (ORFs) within the mRNA (21, 37). In S. cerevisiae, the products of UPF1, UPF2, and UPF3 are required for NMD and provide a surveillance function to lower the abundance of potentially deleterious protein fragments by degrading mRNAs that cannot be translated full length (42). However, the only known growth phenotype of upf mutants is deficient respiration (1). Interestingly, NMD also controls the expression of some wild-type genes. By using high-density oligonucleotide arrays (HDOAs), several hundred wild-type S. cerevisiae mRNAs with either increased or d...

The H-NS protein is a major component of the Escherichia coli nucleoid. Mutations in hns, the gene encoding H-NS, have pleiotropic effects on the cell altering both the expression of a variety of unlinked genes and the inversion rate of the DNA element containing the fimA promoter. We investigated the interaction between H-NS and fimB, the gene encoding the bidirectional recombinase that catalyzes fimA promoter flipping. In ␤-galactosidase assays, we found that fimB expression increased approximately fivefold in an hns2-tetR insertion mutant. In gel mobility shift assays with purified H-NS, we have also shown that H-NS bound directly and cooperatively to the fimB promoter region with greater affinity than for any other known H-NS-regulated gene. Furthermore, this high-affinity interaction resulted in a promoter-specific inhibition of fimB transcription. The addition of purified H-NS to an in vitro transcription system yielded a fivefold or greater reduction in fimB-specific mRNA production. However, the marked increase in cellular FimB levels in the absence of H-NS was not the primary cause of the mutant rapid inversion phenotype. These results are discussed in regard to both H-NS as a transcriptional repressor of fimB expression and its role in regulating type 1 pilus promoter inversion. H-NS is an approximately 15.4-kDa nucleoid-associatedEscherichia coli protein involved in bacterial chromatin condensation (12,49,50). The hns gene is autoregulated (16,54), and its expression is induced three-to fourfold upon cold shock (5, 30). The H-NS protein binds tightly to doublestranded DNA (dsDNA) as a homodimer (15,49). H-NS does not bind DNA in a strict sequence-specific manner (46), yet it preferentially binds curved DNA (43,51,57). Most importantly, H-NS is a global regulator of a variety of unrelated genes in E. coli (2,31,58) as well as being involved in virulence expression in pathogenic Shigella (35), Salmonella (21), and enteroinvasive E. coli (7) strains. In most instances, such as with proU (22, 36), the bgl operon (8,22), and the pap locus (20), mutations in hns cause derepressed gene expression. However, H-NS also acts as a positive regulator for lrp (33) and flagellum biosynthesis genes (3). Two prevailing models exist to explain the role that H-NS plays in modulating gene expression. In the first, H-NS takes an active role by directly binding to DNA and inhibiting transcription (55). In the other, H-NS acts passively in a purely structural role by inducing changes in DNA supercoiling or chromosomal topology (23,25,53).Type 1 pilus expression is predominately controlled at the transcriptional level by the inversion of a 314-bp DNA element containing the promoter of fimA, the gene encoding the major pilus structural subunit (1, 13). Piliation is subject to phase variation whereby in the "ON" orientation, the promoter initiates transcription, FimA monomers are synthesized, assembled, and translocated to the bacterial cell surface. Conversely, when the promoter element is in the "OFF" orientation, fimA trans...

Mutations in hns, the gene encoding the nucleoid-associated protein H-NS, affect both the expression of many specific unlinked genes and the inversion rate of the DNA segment containing the piL4 promoter in Escherichia col. A second-site mutation, termed hscAl, compensated for the effect of an hns-1 mutant allele on the piL4 promoter inversion rate and on activation of the bgl operon. The proU operon, induced in an hns-1 background, remained derepressed in an hns-1 hscAl strain and was induced at an intermediate level in an hns hscAl strain. An insertion mutant allele, hscA2-cat, conferred the same partial hns-l compensatory phenotype as the hscAl allele. The hscA gene encoded a 66-kDa protein product that is a member of the Hsp7O protein class. The gene encoding this product is part of a bicistronic operon that is preceded by a possible r32 promoter and also encodes a 21-kDa protein with significant homology to the DnaJ protein family. The mutation defining the hscAl allele resulted in a phenylalanine substituting a conserved serine residue located in the ATP-binding region of other Hsp7O proteins.H-NS (H1) is an approximately 16-kDa neutral protein that is a major nucleoid component in Escherichia coli (10,35). Homodimers of this protein bind tightly to double-stranded DNA, conferring increased thermal stability to the doublestranded DNA substrate (10,13,35). Although H-NS does not appear to bind DNA in a sequence-dependent fashion (32), this protein has a higher affinity for curved DNA than for DNA substrates with similar sequences that do not possess intrinsic curvature (30,40). H-NS is expressed at high levels in E. coli, as each cell contains approximately 20,000 copies of this protein (35). However, H-NS expression is further induced at least threefold following cold shock (22). The H-NS protein has the additional property of acting to inhibit the expression of a subset of specific genes that are not linked on the E. coli chromosome (3,17,42). Consequently, a number of biological properties can be attributed to this one small protein: it is a part of the prokaryotic nucleoid, it is a stress response protein, and it has an important role in modulating gene expression.Mutations in hns, the gene encoding H-NS, affect gene expression and site-specific DNA inversion in E. coli (7,12,24,36,42), Salmonella strains (15), and Shigella strains (9, 23). In E. coli hns mutants, the inversion rate of the DNA segment containing the pilA promoter is increased over 100-fold (18), the normally cryptic bgl operon is activated (7, 15), the proU operon (normally expressed only under conditions of high osmolarity) is derepressed (15, 24), and Pap pilus expression is also derepressed in strains containing an intact pap locus (12). Different hns mutant alleles display these same phenotypes, but the specific mechanism by which these phenotypes are induced has been the subject of much debate. A number of studies with Salmonella strains have shown that hns mutations result in an increased level of negative supercoiling in reporter...

Hsc66 is the second identified Hsp70 protein in Escherichia coli. Mutations in hscA, the gene encoding Hsc66, compensate for some phenotypic effects of a mutation in hns, a gene encoding the cold-inducible, nucleoidassociated protein H-NS. Expression of hscA was not induced upon heat shock but was induced approximately 11-fold 3 h after a shift from 37 to 10؇C. Furthermore, hscA was induced upon chloramphenicol addition, which induces the synthesis of other cold-inducible genes. Mapping of the transcription initiation site showed that hscA was cotranscribed with an upstream dnaJ-like gene, hscB; thus, hscB was also cold inducible. The hscBA promoter did not contain a Y-box element found in some cold-inducible promoters. Using two-dimensional electrophoresis, we identified Hsc66 under static 37؇C growth conditions and showed that Hsc66 was induced, as well as hscA, 3 h after a cold shock. Growth of an hscA mutant following cold shock was monitored relative to that of an isogenic wild-type strain. While cold shock adaptation as a function of growth rate was not significantly impaired in an hscA mutant, the expression of at least five other proteins was altered in this mutant following cold shock. On the basis of the homology to Hsp70 proteins and the induction following cold shock, we speculate that Hsc66 functions as a cold shock molecular chaperone.Organisms respond to environmental stress by inducing a class of conserved proteins termed heat shock proteins. One subset of these proteins includes the 70-kDa heat shock proteins (Hsp70 proteins). Hsp70 proteins, including the wellcharacterized Escherichia coli DnaK protein (reviewed in reference 2, 9, 10, and 15), function as molecular chaperones assisting unfolded, misfolded, or aggregated proteins in retaining or attaining a specific conformation. Additionally, Hsp70 proteins are involved in protein translocation across cytoplasmic membranes (reviewed in reference 8) and protein degradation (38). The genes encoding Hsp70 proteins are induced following environmental stresses typified by heat shock but including such diverse shocks as ethanol treatment and virus infection.Hsc66, the second Hsp70 protein found in E. coli (23, 35), was identified by screening for mutations that compensated for a specific hns mutation. H-NS is a small, dimeric, nucleoidassociated protein in E. coli (reviewed in reference 43) whose expression can increase from approximately 20,000 copies under static 37ЊC growth (37) to three to four times that amount 3 h following a temperature shift from 37 to 10ЊC (25); thus, H-NS functions as a stress response protein.Shifting growing E. coli from 37 to 10ЊC immediately inhibits cellular growth and protein synthesis. To compensate for this stress, a protein subset is induced between 1 and 4 h following the temperature shift; this induction is termed the cold shock response (reviewed in reference 20). The cold shock response is analogous to the well-characterized heat shock response in that a temperature shift results in cellular stress and protein ...

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In Saccharomyces cerevisiae, Upf3p is required for nonsense-mediated mRNA decay (NMD). Although localized primarily in the cytoplasm, Upf3p contains three sequence elements that resemble nuclear localization signals (NLSs) and two sequence elements that resemble nuclear export signals (NESs). We found that a cytoplasmic reporter protein localized to the nucleus when fused to any one of the three NLS-like sequences of Upf3p. A nuclear reporter protein localized to the cytoplasm when fused to one of the NES-like sequences (NES-A). We present evidence that NES-A functions to signal the export of Upf3p from the nucleus. Combined alanine substitutions in the NES-A element caused a re-distribution of Upf3p to a subnuclear location identified as the nucleolus and conferred an Nmd- phenotype. Single mutations in NES-A failed to affect the distribution of Upf3p and were Nmd+. When an NES element from HIV-1 Rev was inserted near the C terminus of a mutant Upf3p containing multiple mutations in NES-A, the cytoplasmic distribution typical of wild-type Upf3p was restored but the cells remained phenotypically Nmd-. These results suggest that NES-A is a functional nuclear export signal. Combined mutations in NES-A may cause multiple defects in protein function leading to an Nmd- phenotype even when export is restored.