Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents

A dynamic programming algorithm for prediction of RNA secondary structure has been revised to accommodate folding constraints determined by chemical modification and to include free energy increments for coaxial stacking of helices when they are either adjacent or separated by a single mismatch. Furthermore, free energy parameters are revised to account for recent experimental results for terminal mismatches and hairpin, bulge, internal, and multibranch loops. To demonstrate the applicability of this method, in vivo modification was performed on 5S rRNA in both Escherichia coli and Candida albicans with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, dimethyl sulfate, and kethoxal. The percentage of known base pairs in the predicted structure increased from 26.3% to 86.8% for the E. coli sequence by using modification constraints. For C. albicans, the accuracy remained 87.5% both with and without modification data. On average, for these sequences and a set of 14 sequences with known secondary structure and chemical modification data taken from the literature, accuracy improves from 67% to 76%. This enhancement primarily reflects improvement for three sequences that are predicted with <40% accuracy on the basis of energetics alone. For these sequences, inclusion of chemical modification constraints improves the average accuracy from 28% to 78%. For the 11 sequences with <6% pseudoknotted base pairs, structures predicted with constraints from chemical modification contain on average 84% of known canonical base pairs.

https://www.pnas.org/content/pnas/101/19/7287.full.pdf

Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure

Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress. GW bodies/processing bodies (PBs) are distinct cytoplasmic sites of mRNA degradation. In this study, we show that SGs and PBs are spatially, compositionally, and functionally linked. SGs and PBs are induced by stress, but SG assembly requires eIF2alpha phosphorylation, whereas PB assembly does not. They are also dispersed by inhibitors of translational elongation and share several protein components, including Fas-activated serine/threonine phosphoprotein, XRN1, eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, eIF4G, and PABP-1 are restricted to SGs, whereas DCP1a and 2 are confined to PBs. SGs and PBs also can harbor the same species of mRNA and physically associate with one another in vivo, an interaction that is promoted by the related mRNA decay factors TTP and BRF1. We propose that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation.

/pdf/stress-granules-and-processing-bodies-are-dynamically-linked-1jer4cbznv.pdf

Stress granules and processing bodies are dynamically linked sites of mRNP remodeling

mRNA stability in mammalian cells.

Turnover of mRNA is a key mechanism in regulated gene expression. In addition to turnover pathways for normal transcripts, there are surveillance mechanisms that degrade aberrant mRNAs. mRNA decay is regulated in response to cellular signals and coordinated with other mRNA-metabolic processes. When considering the control of gene expression, the focus has traditionally been on transcriptional regulation. Recently, however, the large contribution made by mRNA decay has become difficult to ignore. Large-scale analyses indicate that as many as half of all changes in the amounts of mRNA in some responses can be attributed to altered rates of decay. In this article, we discuss some of the mechanisms that are used by the cell to mediate and regulate this intriguing process.

The highways and byways of mRNA decay.

Disruption of the gene XRN1, coding for a 5'----3' exoribonuclease, restricts yeast cell growth.

Four new small polypeptides are associated with DNA-dependent RNA polymerase from E. coli after infection with T4 phage. The new polypeptides are easily detected in RNA polymerase from E. coli cells labeled with amino acids after phage infection. Their molecular weights range from 10,000 to 22,000, as detected by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All four polypeptides are found after infection with either wild-type T4 phage or T4 early amber mutants in genes 44, 42, 47, and 46. None of the polypeptides is labeled significantly before 5 min after infection at 30°. When two maturation-defective amber mutants in gene 55 of T4 phage are used for infection, a polypeptide with a molecular weight of 22,000 is absent. When a maturation-defective amber mutant in gene 33 of T4 phage is used, another small protein is absent.

http://www.pnas.org/content/69/3/603.full.pdf

New small polypeptides associated with DNA-dependent RNA polymerase of Escherichia coli after infection with bacteriophage T4.

Characterization of the XRN1 gene encoding a 5'-->3' exoribonuclease: sequence data and analysis of disparate protein and mRNA levels of gene-disrupted yeast cells.

The portion of the internal transcribed spacer 1 found on 20S pre-rRNA accumulates in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1, showing that an endonucleolytic cleavage at the 3' terminus of 18S rRNA is involved in the 20S pre-rRNA to 18S mature rRNA conversion. Smaller fragments of the spacer sequence are also found. The exoribonuclease may be involved as a cytoplasmic RNase in the hydrolysis of the spacer.

Audrey Stevens

Papers

Disruption of the gene XRN1, coding for a 5'----3' exoribonuclease, restricts yeast cell growth.

New small polypeptides associated with DNA-dependent RNA polymerase of Escherichia coli after infection with bacteriophage T4.

Characterization of the XRN1 gene encoding a 5'-->3' exoribonuclease: sequence data and analysis of disparate protein and mRNA levels of gene-disrupted yeast cells.

Fragments of the internal transcribed spacer 1 of pre-rRNA accumulate in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1.

5'-exonuclease-2 of Saccharomyces cerevisiae. Purification and features of ribonuclease activity with comparison to 5'-exonuclease-1.