A New Yeast Poly(A) Polymerase Complex Involved in RNA Quality Control

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNA(Met) (tRNA(i) (Met)). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNA(i) (Met) with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNA(i) (Met) by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover

Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control

Trf4 is the poly(A) polymerase component of TRAMP4, which stimulates nuclear RNA degradation by the exosome. We report that in Saccharomyces cerevisiae strains lacking Trf4, cryptic transcripts are detected from regions of repressed chromatin at telomeres and the rDNA intergenic spacer region (IGS1-R), and at CEN3. Degradation of the IGS1-R transcript was reduced in strains lacking TRAMP components, the core exosome protein Mtr3 or the nuclear-specific exosome component Rrp6. IGS1-R has potential binding sites for the RNA-binding proteins Nrd1/Nab3, and was stabilized by mutation of Nrd1. IGS1-R passes through the replication fork barrier, a region required for rDNA copy number control. Strains lacking Trf4 showed sporadic changes in rDNA copy number, whereas loss of both Trf4 and either the histone deacetylase Sir2 or the topoisomerase Top1 caused dramatic loss of rDNA repeats. Chromatin immunoprecipitation analyses showed that Trf4 is co-transcriptionally recruited to IGS1-R, consistent with a direct role in rDNA stability. Co-transcriptional RNA binding by Trf4 may link RNA and DNA metabolism and direct immediate IGS1-R degradation by the exosome following transcription termination

A Technical and Economic Evaluation of Geosynthetic Rolled Erosion Control Products in Highway Drainage Channels

Historically, local village, town, and county highway departments have relied heavily on the use of stone fill and rock riprap to line highway drainage channels. These are often constructed without the benefit of design or the evaluation of alternatives, because they have always been done this way. In September 1999, the Munro Road reconstruction project was completed in Onondaga County, New York. As part of an erosion and sediment control demonstration project, the drainage portion of the project was redesigned using geosynthetic rolled erosion control products (RECPs) in lieu of stone fill and rock riprap on almost 1,130 m of channel. The use of the RECPs saved approximately $95,800 in construction costs and is expected to lower maintenance costs as well as provide long-term protection against erosion. The overall purpose of the project was to demonstrate to highway departments the functional and economical advantages of using RECPs for these applications. This paper summarizes the design, construction, and performance to date of seven of the ten different RECPs installed for the project. </jats:p

Cytoplasmic poly(A) polymerases mediate cellular responses to S phase arrest

The S-M checkpoint delays mitosis until DNA replication is complete; cells defective in this checkpoint lose viability when DNA replication is inhibited. This inviability can be suppressed in fission yeast by overexpression of Cid1 or the related protein Cid13. Fission yeast contain six cid1/cid13-like genes, whereas budding yeast has just two, TRF4 and TRF5. Trf4 and Trf5 were recently reported to comprise an essential DNA polymerase activity required for the establishment of sister chromatid cohesion. In contrast, we find that Cid1 is not a DNA polymerase but instead uses RNA substrates and has poly(A) polymerase activity. Unlike the previously characterized yeast poly(A) polymerase, which is a nuclear enzyme, Cid1 and Cid13 are constitutively cytoplasmic. Cid1 has a degree of substrate specificity in vitro, consistent with the notion that it targets a subset of cytoplasmic mRNAs for polyadenylation in vivo, hence increasing their stability and/or efficiency of translation. Preferred Cid1 targets presumably include mRNAs encoding components of the S-M checkpoint, whereas Cid13 targets are likely to be involved in dNTP metabolism. Cytoplasmic polyadenylation is known to be an important regulatory mechanism during early development in animals. Our findings in yeast suggest that this level of gene regulation is of more general significance in eukaryotic cells

Roles for cytoplasmic polyadenylation in cell cycle regulation

Polyadenylation of eukaryotic mRNAs in the nucleus promotes their translation following export to the cytoplasm and is an important determinant of mRNA stability. An additional level of control of gene expression is provided by cytoplasmic polyadenylation, which activates translation of a number of mRNAs important in orchestrating cell cycle events in oocytes. Recent studies indicate that cytoplasmic polyadenylation may be a mechanism of translational activation that is more widespread in eukaryotic cells. Here we discuss the roles of a recently identified family of nucleotidyl transferases (encoded by the cid1 gene family) in cell cycle regulation. To date, this family has been characterised mainly in yeasts, but it is conserved throughout the eukaryotes. Biochemical studies have indicated that a subset of members of this family function as cytoplasmic poly(A) polymerases targeting specific mRNAs for translation. This form of translational control appears to be particularly important for cell cycle regulation following inhibition of DNA synthesis