KSRP-PMR1-exosome association determines parathyroid hormone mRNA levels and stability in transfected cells

<p>Abstract</p> <p>Background</p> <p>Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally through the binding of the <it>trans-</it>acting proteins AU rich binding factor 1 (AUF1), Upstream of N-<it>ras </it>(Unr) and KH-type splicing regulatory protein (KSRP) to an AU rich element (ARE) in PTH mRNA 3'-UTR. AUF1 and Unr stabilize PTH mRNA while KSRP, recruiting the exoribonucleolytic complex exosome, promotes PTH mRNA decay.</p> <p>Results</p> <p>PTH mRNA is cleaved by the endoribonuclease polysomal ribonuclease 1 (PMR1) in an ARE-dependent manner. Moreover, PMR1 co-immunoprecipitates with PTH mRNA, the exosome and KSRP. Knock-down of either exosome components or KSRP by siRNAs prevents PMR1-mediated cleavage of PTH mRNA.</p> <p>Conclusion</p> <p>PTH mRNA is a target for the endonuclease PMR1. The PMR1 mediated decrease in PTH mRNA levels involves the PTH mRNA 3'-UTR ARE, KSRP and the exosome. This represents an unanticipated mechanism by which the decay of an ARE-containing mRNA is facilitated by KSRP and is dependent on both the exosome and an endoribonuclease.</p

An Evolutionary Analysis of Lateral Gene Transfer in Thymidylate Synthase Enzymes

Thymidylate synthases (Thy) are key enzymes in the synthesis of deoxythymidylate, 1 of the 4 building blocks of DNA. As such, they are essential for all DNA-based forms of life and therefore implicated in the hypothesized transition from RNA genomes to DNA genomes. Two evolutionally unrelated Thy enzymes, ThyA and ThyX, are known to catalyze the same biochemical reaction. Both enzymes are sporadically distributed within each of the 3 domains of life in a pattern that suggests multiple nonhomologous lateral gene transfer (LGT) events. We present a phylogenetic analysis of the evolution of the 2 enzymes, aimed at unraveling their entangled evolutionary history and tracing their origin back to early life. A novel probabilistic evolutionary model was developed, which allowed us to compute the posterior probabilities and the posterior expectation of the number of LGT events. Simulation studies were performed to validate the model's ability to accurately detect LGT events, which have occurred throughout a large phylogeny. Applying the model to the Thy data revealed widespread nonhomologous LGT between and within all 3 domains of life. By reconstructing the ThyA and ThyX gene trees, the most likely donor of each LGT event was inferred. The role of viruses in LGT of Thy is finally discussed

Deep Panning: Steps towards Probing the IgOme

Background: Polyclonal serum consists of vast collections of antibodies, products of differentiated B-cells. The spectrum of antibody specificities is dynamic and varies with age, physiology, and exposure to pathological insults. The complete repertoire of antibody specificities in blood, the IgOme, is therefore an extraordinarily rich source of information–a molecular record of previous encounters as well as a status report of current immune activity. The ability to profile antibody specificities of polyclonal serum at exceptionally high resolution has been an important and serious challenge which can now be overcome. Methodology/Principal Findings: Here we illustrate the application of Deep Panning, a method that combines the flexibility of combinatorial phage display of random peptides with the power of high-throughput deep sequencing. Deep Panning is first applied to evaluate the quality and diversity of naïve random peptide libraries. The production of very large data sets, hundreds of thousands of peptides, has revealed unexpected properties of combinatorial random peptide libraries and indicates correctives to ensure the quality of the libraries generated. Next, Deep Panning is used to analyze a model monoclonal antibody in addition to allowing one to follow the dynamics of biopanning and peptide selection. Finally Deep Panning is applied to profile polyclonal sera derived from HIV infected individuals. Conclusions/Significance: The ability to generate and characterize hundreds of thousands of affinity-selected peptide

Pie charts depicting the proportion of unique peptides in phage display libraries.

<p>A total of 155,241 inserts were read for the random phage display peptide library (<b>A</b>). 24% of the peptides contained at least one UAA or UGA stop codon (red plus dark red). 58% of the peptides were unique containing a UAG stop codon (light green) of these some exist in multiple copies (3% of the total, dark green). 15% of the peptides were completely devoid of stop codons (blue, less than 1% had 2–5 copies). Pie Chart (<b>B</b>) depicts the same set of peptides devoid of all those that had detectable frameshifted inserts (37,223 inserts leaving 118,018 functional peptides of which ca 1% contained stop codons UAA and UGA nonetheless). A second library was constructed in DH5alpha <i>supE144</i> cells (<b>C</b>). Values below 1% are not given.</p

Three rounds of panning with mAb GV4H3.

<p>GV4H3 mAb was used to bio-pan the 7 mer random peptide library 3 consecutive rounds of panning (Capture #1 through #3) and compared with the naïve library. For each sample the 20 top most frequent peptides are given along with the number of times they appear. The number of unique versus total peptides is shown as well. Numbers in parentheses represent the percent value of the total peptides for each category. Bold sequences indicate peptides that are carried over from Capture #1. Bold and Italic sequences indicate peptides carried over from Capture #2.</p

Deep Panning with mAb GV4H3.

<p>(<b>A</b>) Mapitope prediction of the GV4H3 epitope on HIV gp120. The top 20 peptides of Capture #2 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041469#pone-0041469-t001" target="_blank">Table 1</a>) were used as the dataset for Mapitope prediction of the GV4H3 epitope. The single predicted cluster comprises two discontinuous segments of the antigen (green and blue) brought to flank the core of the epitope (residues 221–226, pink). (<b>B-E</b>) MEME analysis of the GV4H3 derived peptides. The 20 top most frequent peptides of Capture #3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041469#pone-0041469-t001" target="_blank">Table 1</a>) generated a major motif “AGWAV”. This motif (<b>B</b>) and three additional motifs are identified when all 4,823 peptides are analyzed. The “VGF” motif (<b>C</b>) is a simpler version of the major motif. The two additional minor motifs (<b>D</b> and <b>E</b>) do not have obvious similarity to the epitope of the mAb. The “ADGIGGG” motif clearly corresponds with the most frequent peptide ADGIVGW (see text). The numbers in red represent the number of unique peptides that define each motif.</p

BLASTP analysis of HIVIG-captured peptides against viral coat proteins.

<p>Of the 223 top unique HIVIG-captured peptides, 18 (8%) scored hits in BLASTP analysis against the HIV-1_HXB2 gp160 (blue). Repeating this procedure with the same protein but scrambled gives an average value of 5.5 hits when performed 1,000 times (2.5%±2.2 s.d., red). The differences between native and scrambled coat proteins BLASTP results of 11 other RNA viruses were not found to be significant. <i>*P</i><0.01.</p