A Computational Pipeline for High- Throughput Discovery of cis-Regulatory Noncoding RNA in Prokaryotes

Abstract

Noncoding RNAs (ncRNAs) are important functional RNAs that do not code for proteins. We present a highly efficient computational pipeline for discovering cis-regulatory ncRNA motifs de novo. The pipeline differs from previous methods in that it is structure-oriented, does not require a multiple-sequence alignment as input, and is capable of detecting RNA motifs with low sequence conservation. We also integrate RNA motif prediction with RNA homolog search, which improves the quality of the RNA motifs significantly. Here, we report the results of applying this pipeline to Firmicute bacteria. Our top-ranking motifs include most known Firmicute elements found in the RNA family database (Rfam). Comparing our motif models with Rfam's hand-curated motif models, we achieve high accuracy in both membership prediction and base-pair–level secondary structure prediction (at least 75% average sensitivity and specificity on both tasks). Of the ncRNA candidates not in Rfam, we find compelling evidence that some of them are functional, and analyze several potential ribosomal protein leaders in depth.

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA

“…Sequences with a 5 ′ UTR shorter than 40 nt were removed, and those longer than 400 nt were limited to 400 residues at the 5 ′ end. The 5 ′ -UTR sequences were searched for conserved RNA motifs with CMfinder (Yao et al 2007), a local RNA sequence alignment algorithm based on covariance models. The program was started using two parameter sets: (1) returning up to five candidate motifs of length 30-100 nt and containing one stem-loop; and (2) returning up to five motifs of length 40-100 nt and containing two stem-loops.…”

“…Overlapping motifs were subsequently combined using the CombMotif.pl script from the CMfinder package. To favor thermostable motifs present in many genomes, with conserved sequence and secondary structure, for each of the motifs a score was assigned according to the heuristic function proposed by Yao et al (2007). The best-scored motif was used to calculate a covariance model, subsequently used to scan the entire genome sequences with cmsearch ) and e-value threshold of 0.01.…”

“…However, no mechanism regulating 3 their expression has been proposed so far. In a high-throughput bioinformatics screening study, the Breaker group identified a conserved RNA motif associated with rpsF (Yao et al 2007), suggesting presence of a regulatory mechanism. However, this study focused only on Firmicutes bacteria and did not elaborate on the structure or the potential role of this motif.…”

Bacterial RNA motif in the 5′ UTR of rpsF interacts with an S6:S18 complex

“…In the past few years, increasingly sophisticated comparative genomic approaches have predicted many putative RNA structures within bacterial genomes (Barrick et al 2004;Weinberg et al 2007Weinberg et al , 2010Yao et al 2007;Meyer et al 2009;Xu et al 2009). Many of these RNAs have been experimentally characterized as riboswitches (for review, see Serganov and Nudler 2013), or other functional RNAs (Irnov and Winkler 2010).…”

Current knowledge on regulatory RNAs and their machineries inStaphylococcus aureus

“…This specific interaction often induces mRNA conformational changes that have direct consequences on the expression of the following coding sequence. 40 Based on sequence and structure conservation, 41,42 15 riboswitches were mapped on S. aureus genome. Until now, 7 operons and 33 genes are expected to be under the control of riboswitches specific for S-adenosylmethionine (SAM), thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), lysine, glycine, guanine, 7-aminomethyl-7-deazaguanine (preQ1) and glucosamine-6-phosphate (Glc-6P).…”