Project description:Poly(A) tails are important elements in mRNA translation and stability, although recent genome-wide studies have concluded that poly(A) tail length is generally not associated with translational efficiency in nonembryonic cells. To investigate whether poly(A) tail size might be coupled to gene expression in an intact organism, we used an adapted TAIL-seq protocol to measure poly(A) tails in Caenorhabditis elegans. Surprisingly, we found that well-expressed transcripts contain relatively short, well-defined tails. This attribute appears to be dependent on translational efficiency, as transcripts enriched for optimal codons and ribosome association had the shortest tail sizes, whereas noncoding RNAs retained long tails. Across eukaryotes, short tails were a feature of abundant and well-translated mRNAs. This seems to contradict the dogma that deadenylation induces translational inhibition and mRNA decay and suggests that well-expressed mRNAs accumulate with pruned tails that accommodate a minimal number of poly(A)-binding proteins, which may be ideal for protective and translational functions.
Project description:It is not always easy to apply microarray technology to small numbers of cells because of the difficulty in selectively isolating mRNA from such cells. We report here the preparation of mRNA from ciliated sensory neurons of Caenorhabditis elegans using the mRNA-tagging method, in which poly(A) RNA was co-immunoprecipitated with an epitope-tagged poly(A)-binding protein specifically expressed in sensory neurons. Subsequent cDNA microarray analyses led to the identification of a panel of sensory neuron-expressed genes.
Project description:BackgroundThe Poly(A) effect is a cross-hybridization artifact in which poly(T)-containing molecules, which are produced by the reverse transcription of a poly(A)+ RNA mixture, bind promiscuously to the poly(A) stretches of the DNA in microarray spots. It is customary to attempt to block such hybridization by adding poly(A) to the hybridization solution. This note describes an experiment intended to evaluate circumstances under which the blocking procedure may not have been successful.ResultsThe experiment involves a spot-by-spot comparison between the hybridization signals obtained by hybridizing a microarray to: (1) end-labeled oligo(dT), versus, (2) cDNA prepared from muscle tissue. We found that the blocking appears to be successful for the vast majority of microarray spots, as evidenced by the weakness of the correlation between signals (1) and (2). However, we found that for microarray spots having oligo(dT) hybridization levels greater than a certain threshold, the blocking might be ineffective or incomplete, as evidenced by an exceptionally strong signal (2) whenever signal (1) is greater than the threshold.ConclusionThe PolyA effect may be more subtle than simply a hybridization signal that is proportional to the PolyA content of each microarray spot. It may instead be present only in spots that hybridize oligo(dT) greater than some threshold level. The strong signal generated at these "outlier" spots by cDNA probes might be due to the formation of hybridization heteropolymers.
Project description:BackgroundIn animals, the moss Physcomitrella patens and the pollen of Arabidopsis thaliana, highly expressed genes have shorter introns than weakly expressed genes. A popular explanation for this is selection for transcription efficiency, which includes two sub-hypotheses: to minimize the energetic cost or to minimize the time cost.ResultsIn an individual human, different organs may differ up to hundreds of times in cell number (for example, a liver versus a hypothalamus). Considered at the individual level, a gene specifically expressed in a large organ is actually transcribed tens or hundreds of times more than a gene with a similar expression level (a measure of mRNA abundance per cell) specifically expressed in a small organ. According to the energetic cost hypothesis, the former should have shorter introns than the latter. However, in humans and mice we have not found significant differences in intron length between large-tissue/organ-specific genes and small-tissue/organ-specific genes with similar expression levels. Qualitative estimation shows that the deleterious effect (that is, the energetic burden) of long introns in highly expressed genes is too negligible to be efficiently selected against in mammals.ConclusionThe short introns in highly expressed genes should not be attributed to energy constraint. We evaluated evidence for the time cost hypothesis and other alternatives.
Project description:Small non-coding RNAs (sncRNAs) are a class of transcripts implicated in several eukaryotic regulatory mechanisms, namely gene silencing and chromatin regulation. Despite significant progress in their identification by next generation sequencing (NGS) we are still far from understanding their full diversity and functional repertoire.Here we report the identification of tRNA derived fragments (tRFs) by NGS of the sncRNA fraction of zebrafish. The tRFs identified are 18-30 nt long, are derived from specific 5' and 3' processing of mature tRNAs and are differentially expressed during development and in differentiated tissues, suggesting that they are likely produced by specific processing rather than random degradation of tRNAs. We further show that a highly expressed tRF (5'tRF-Pro(CGG)) is cleaved in vitro by Dicer and has silencing ability, indicating that it can enter the RNAi pathway. A computational analysis of zebrafish tRFs shows that they are conserved among vertebrates and mining of publicly available datasets reveals that some 5'tRFs are differentially expressed in disease conditions, namely during infection and colorectal cancer.tRFs constitute a class of conserved regulatory RNAs in vertebrates and may be involved in mechanisms of genome regulation and in some diseases.
Project description:Based primarily on 16S rRNA sequence comparisons, life has been broadly divided into the three domains of Bacteria, Archaea, and Eukarya. Archaea is further classified into Crenarchaea and Euryarchaea. Archaea generally thrive in extreme environments as assessed by temperature, pH, and salinity. For many prokaryotic organisms, ribosomal proteins (RP), transcription/translation factors, and chaperone genes tend to be highly expressed. A gene is predicted highly expressed (PHX) if its codon usage is rather similar to the average codon usage of at least one of the RP, transcription/translation factors, and chaperone gene classes and deviates strongly from the average gene of the genome. The thermosome (Ths) chaperonin family represents the most salient PHX genes among Archaea. The chaperones Trigger factor and HSP70 have overlapping functions in the folding process, but both of these proteins are lacking in most archaea where they may be substituted by the chaperone prefoldin. Other distinctive PHX proteins of Archaea, absent from Bacteria, include the proliferating cell nuclear antigen PCNA, a replication auxiliary factor responsible for tethering the catalytic unit of DNA polymerase to DNA during high-speed replication, and the acidic RP P0, which helps to initiate mRNA translation at the ribosome. Other PHX genes feature Cell division control protein 48 (Cdc48), whereas the bacterial septation proteins FtsZ and minD are lacking in Crenarchaea. RadA is a major DNA repair and recombination protein of Archaea. Archaeal genomes feature a strong Shine-Dalgarno ribosome-binding motif more pronounced in Euryarchaea compared with Crenarchaea.