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Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting.

ABSTRACT: So far, the annotation of translation initiation sites (TISs) has been based mostly upon bioinformatics rather than experimental evidence. We adapted ribosomal footprinting to puromycin-treated cells to generate a transcriptome-wide map of TISs in a human monocytic cell line. A neural network was trained on the ribosomal footprints observed at previously annotated AUG translation initiation codons (TICs), and used for the ab initio prediction of TISs in 5062 transcripts with sufficient sequence coverage. Functional interpretation suggested 2994 novel upstream open reading frames (uORFs) in the 5' UTR, 1406 uORFs overlapping with the coding sequence, and 546 N-terminal protein extensions. The TIS detection method was validated on the basis of previously published alternative TISs and uORFs. Among primates, TICs in newly annotated TISs were significantly more conserved than control codons, both for AUGs and near-cognate codons. The transcriptome-wide map of novel candidate TISs derived as part of the study will shed further light on the way in which human proteome diversity is influenced by alternative translation initiation and regulation.

SUBMITTER: Fritsch C

PROVIDER: S-EPMC3483550 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting.

Fritsch Claudia C Herrmann Alexander A Nothnagel Michael M Szafranski Karol K Huse Klaus K Schumann Frank F Schreiber Stefan S Platzer Matthias M Krawczak Michael M Hampe Jochen J Brosch Mario M

Genome research 20120809 11

So far, the annotation of translation initiation sites (TISs) has been based mostly upon bioinformatics rather than experimental evidence. We adapted ribosomal footprinting to puromycin-treated cells to generate a transcriptome-wide map of TISs in a human monocytic cell line. A neural network was trained on the ribosomal footprints observed at previously annotated AUG translation initiation codons (TICs), and used for the ab initio prediction of TISs in 5062 transcripts with sufficient sequence ...[more]

PMID: 22879431

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Project description:BackgroundSmall RNAs (sRNAs) are widespread among bacteria and have diverse regulatory roles. Most of these sRNAs have been discovered by a combination of computational and experimental methods. In Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium and opportunistic human pathogen, the GacS/GacA two-component system positively controls the transcription of two sRNAs (RsmY, RsmZ), which are crucial for the expression of genes involved in virulence. In the biocontrol bacterium Pseudomonas fluorescens CHA0, three GacA-controlled sRNAs (RsmX, RsmY, RsmZ) regulate the response to oxidative stress and the expression of extracellular products including biocontrol factors. RsmX, RsmY and RsmZ contain multiple unpaired GGA motifs and control the expression of target mRNAs at the translational level, by sequestration of translational repressor proteins of the RsmA family.ResultsA combined computational and experimental approach enabled us to identify 14 intergenic regions encoding sRNAs in P. aeruginosa. Eight of these regions encode newly identified sRNAs. The intergenic region 1698 was found to specify a novel GacA-controlled sRNA termed RgsA. GacA regulation appeared to be indirect. In P. fluorescens CHA0, an RgsA homolog was also expressed under positive GacA control. This 120-nt sRNA contained a single GGA motif and, unlike RsmX, RsmY and RsmZ, was unable to derepress translation of the hcnA gene (involved in the biosynthesis of the biocontrol factor hydrogen cyanide), but contributed to the bacterium's resistance to hydrogen peroxide. In both P. aeruginosa and P. fluorescens the stress sigma factor RpoS was essential for RgsA expression.ConclusionThe discovery of an additional sRNA expressed under GacA control in two Pseudomonas species highlights the complexity of this global regulatory system and suggests that the mode of action of GacA control may be more elaborate than previously suspected. Our results also confirm that several GGA motifs are required in an sRNA for sequestration of the RsmA protein.

Dataset Information

Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting.

Publications

Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting.

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