Identification of bacterial sRNA regulatory targets using ribosome profiling

Bacteria express large numbers of non-coding, regulatory RNAs known as ‘small RNAs’ (sRNAs). sRNAs typically regulate expression of multiple target messenger RNAs (mRNAs) through base-pairing interactions. sRNA:mRNA base-pairing often results in altered mRNA stability and/or altered translation initiation. Computational identification of sRNA targets is challenging due to the requirement for only short regions of base-pairing that can accommodate mismatches. Experimental approaches have been applied to identify sRNA targets on a genomic scale, but these focus only on those targets regulated at the level of mRNA stability. Here, we utilize ribosome profiling (Ribo-seq) to experimentally identify regulatory targets of the Escherichia coli sRNA RyhB. We not only validate a majority of known RyhB targets using the Ribo-seq approach, but also discover many novel ones. We further confirm regulation of a selection of known and novel targets using targeted reporter assays. By mutating nucleotides in the mRNA of a newly discovered target, we demonstrate direct regulation of this target by RyhB. Moreover, we show that Ribo-seq distinguishes between mRNAs regulated at the level of RNA stability and those regulated at the level of translation. Thus, Ribo-seq represents a powerful approach for genome-scale identification of sRNA targets.     

The DHX33 RNA Helicase Promotes mRNA Translation Initiation

DEAD/DEAH box RNA helicases play essential roles in numerous RNA metabolic processes, such as mRNA translation, pre-mRNA splicing, ribosome biogenesis, and double-stranded RNA sensing. Herein we show that a recently characterized DEAD/DEAH box RNA helicase, DHX33, promotes mRNA translation initiation. We isolated intact DHX33 protein/RNA complexes in cells and identified several ribosomal proteins, translation factors, and mRNAs. Reduction of DHX33 protein levels markedly reduced polyribosome formation and caused the global inhibition of mRNA translation that was rescued with wild-type DHX33 but not helicase-defective DHX33. Moreover, we observed an accumulation of mRNA complexes with the 80S ribosome in the absence of functional DHX33, consistent with a stalling in initiation, and DHX33 more preferentially promoted structured mRNA translation. We conclude that DHX33 functions to promote elongation-competent 80S ribosome assembly at the late stage of mRNA translation initiation. Our results reveal a newly recognized function of DHX33 in mRNA translation initiation, further solidifying its central role in promoting cell growth and proliferation.     

ADAR-mediated RNA editing in non-coding RNA sequences

Adenosine to inosine (A-to-I) RNA editing is the most abundant editing event in animals. It converts adenosine to inosine in double-stranded RNA regions through the action of the adenosine deaminase acting on RNA (ADAR) proteins. Editing of pre-mRNA coding regions can alter the protein codon and increase functional diversity. However, most of the A-to-I editing sites occur in the non-coding regions of pre-mRNA or mRNA and non-coding RNAs. Untranslated regions (UTRs) and introns are located in pre-mRNA non-coding regions, thus A-to-I editing can influence gene expression by nuclear retention, degradation, alternative splicing, and translation regulation. Non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA) and long non-coding RNA (lncRNA) are related to pre-mRNA splicing, translation, and gene regulation. A-to-I editing could therefore affect the stability, biogenesis, and target recognition of non-coding RNAs. Finally, it may influence the function of non-coding RNAs, resulting in regulation of gene expression. This review focuses on the function of ADAR-mediated RNA editing on mRNA non-coding regions (UTRs and introns) and non-coding RNAs (miRNA, siRNA, and lncRNA).     

An atlas and analysis of bovine skeletal muscle long noncoding RNAs

Long noncoding RNAs (lncRNAs) have various biological functions and have been extensively studied in recent years. However, the identification and characterization of bovine lncRNAs in skeletal muscle has been very limited compared with that of lncRNAs in other model organisms. In this study, 7188 bovine skeletal muscle lncRNAs were identified by RNA‐Seq and a stringent screening procedure in four different muscle tissues. These lncRNAs shared many characteristics with other mammalian lncRNAs, such as a shorter open reading frame and lower expression level than for mRNAs. Furthermore, the chromosomal locations and global expression patterns for these lncRNAs are also described in detail. More importantly, we detected the important interaction relationships of lncRNAs–miRNAs–mRNAs related to muscle development among 36 lncRNAs, 62 miRNAs and 12 mRNAs. Our results provide a global expression pattern of lncRNAs specific to bovine skeletal muscle and provide important targets for revealing the function of bovine muscle development by thoroughly studying the interaction relationships of lncRNAs–miRNAs–mRNAs.     

From microRNAs to targets: pathway discovery in cell fate transitions

MicroRNAs (miRNAs) are 22 nt non-coding RNAs that regulate expression of downstream targets by messenger RNA (mRNA) destabilization and translational inhibition. A large number of eukaryotic mRNAs are targeted by miRNAs, with many individual mRNAs being targeted by multiple miRNAs. Further, a single miRNA can target hundreds of mRNAs, making these small RNAs powerful regulators of cell fate decisions. Such regulation by miRNAs has been observed in the maintenance of the embryonic stem cell (ESC) cell cycle and during ESC differentiation. MiRNAs can also promote the dedifferentiation of somatic cells to induced pluripotent stem cells. During this process they target multiple downstream genes, which represent important nodes of key cellular processes. Here, we review these findings and discuss how miRNAs may be used as tools to discover novel pathways that are involved in cell fate transitions using dedifferentiation of somatic cells to induced pluripotent stem cells as a case study.     

Specificity of mRNA Folding and Its Association with Evolutionarily Adaptive mRNA Secondary Structures

The secondary structure is a fundamental feature of both non-coding RNAs (ncRNAs) and messenger RNAs (mRNAs). However, our understanding of the secondary structures of mRNAs, especially those of the coding regions, remains elusive, likely due to translation and the lack of RNA-binding proteins that sustain the consensus structure like those binding to ncRNAs. Indeed, mRNAs have recently been found to adopt diverse alternative structures, but the overall functional significance remains untested. We hereby approach this problem by estimating the folding specificity, i.e., the probability that a fragment of an mRNA folds back to the same partner once refolded. We show that the folding specificity of mRNAs is lower than that of ncRNAs and exhibits moderate evolutionary conservation. Notably, we find that specific rather than alternative folding is likely evolutionarily adaptive since specific folding is frequently associated with functionally important genes or sites within a gene. Additional analysis in combination with ribosome density suggests the ability to modulate ribosome movement as one potential functional advantage provided by specific folding. Our findings reveal a novel facet of the RNA structurome with important functional and evolutionary implications and indicate a potential method for distinguishing the mRNA secondary structures maintained by natural selection from molecular noise.     

Expanding roles for miRNAs and siRNAs in cell regulation

The role of small RNAs as key regulators of mRNA turnover and translation has been well established. Recent advances indicate that the small RNAs termed microRNAs play important roles in cell proliferation, apoptosis and differentiation. Moreover, the microRNA mechanism is an efficient means to regulate production of a diverse range of proteins. As new microRNAs and their mRNA targets rapidly emerge, it is becoming apparent that RNA-based regulation of mRNAs may rival ubiquitination as a mechanism to control protein levels.