Relationship between Yeast Polyribosomes and Upf Proteins Required for Nonsense mRNA Decay

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 ly-sates with RNase A caused a coincident collapse of polyribosomes and each Upf protein into frac-tions 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 gradi-ent 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

Relationship between Yeast Polyribosomes and Upf Proteins Required for Nonsense mRNA Decay

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 ly-sates with RNase A caused a coincident collapse of polyribosomes and each Upf protein into frac-tions 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 gradi-ent 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

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

A highly virulent variant of HIV-1 circulating in the Netherlands

We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log10 increase (i.e., a ~3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advanced HIV-CD4 cell counts below 350 cells per cubic millimeter, with long-term clinical consequences-is expected to be reached, on average, 9 months after diagnosis for individuals in their thirties with this variant. Age, sex, suspected mode of transmission, and place of birth for the aforementioned 109 individuals were typical for HIV-positive people in the Netherlands, which suggests that the increased virulence is attributable to the viral strain. Genetic sequence analysis suggests that this variant arose in the 1990s from de novo mutation, not recombination, with increased transmissibility and an unfamiliar molecular mechanism of virulence