Unified translation repression mechanism for microRNAs and upstream AUGs

Abstract

Background: MicroRNAs (miRNAs) are endogenous small RNAs that modulate gene expression at the post-transcriptional level by binding complementary sites in the 3'-UTR. In a recent genome-wide study reporting a new miRNA target class (miBridge), we identified and validated interactions between 5'-UTRs and miRNAs. Separately, upstream AUGs (uAUGs) in 5'-UTRs are known to regulate genes translationally without affecting mRNA levels, one of the mechanisms for miRNA-mediated repression.

Results: Using sequence data from whole-genome cDNA alignments we identified 1418 uAUG sequences on the 5'-UTR that specifically interact with 3'-ends of conserved miRNAs. We computationally identified miRNAs that can target six genes through their uAUGs that were previously reported to suppress translation. We extended this meta-analysis by confirming expression of these miRNAs in cell-lines used in the uAUG studies. Similarly, seven members of the KLF family of genes containing uAUGs were computationally identified as interacting with several miRNAs. Using KLF9 as an example (whose protein expression is limited to brain tissue despite the mRNA being expressed ubiquitously), we show computationally that miRNAs expressed only in HeLa cells and not in neuroblastoma (N2A) cells can bind the uAUGs responsible for translation inhibition. Our computed results demonstrate that tissue- or cell-line specific repression of protein translation by uAUGs can be explained by the presence or absence of miRNAs that target these uAUG sequences. We propose that these uAUGs represent a subset of miRNA interaction sites on 5'-UTRs in miBridge, whereby a miRNA binding a uAUG hinders the progression of ribosome scanning the mRNA before it reaches the open reading frame (ORF).

Conclusions: While both miRNAs and uAUGs are separately known to down-regulate protein expression, we show that they may be functionally related by identifying potential interactions through a sequence-specific binding mechanism. Using prior experimental evidence that shows uAUG effects on translation repression together with miRNA expression data specific to cell lines, we demonstrate through computational analysis that cell-specific down-regulation of protein expression (while maintaining mRNA levels) correlates well with the simultaneous presence of miRNA and target uAUG sequences in one cell type and not others, suggesting tissue-specific translation repression by miRNAs through uAUGs.

Figure 1

Number of uAUGs in 5'-UTRs and their conservation. (A) Distribution of uAUGs in human 5'-UTR sequences (B) Fraction of uAUG-containing n-mer sequences conserved in human and mouse 5'-UTRs.

Figure 2

Interaction of miRNAs with uAUG sequences. Each predicted interaction is characterized by a 7-mer consecutive match between the indicated half of mature miRNA (5p and 3p for the 5'- and 3'-end respectively) and uAUG sequence with ΔG₃₇ ≤ -14 kcal mol^-1. Grey bars represent actual counts and white bars represent average number of counts over 1000 repetitions of miRNA shuffling. Error bars represent the standard deviations. Significant outcomes are indicated with the corresponding p-values. (A, B) Number of interactions between uAUG sequences (4009 in total) and conserved and non-conserved miRNAs (471 and 206 in total respectively) without GU wobbles (A) and with at most one GU wobble (B). (C, D) Number of interactions between conserved miRNAs and uAUG sequences (2935 conserved and 1074 non-conserved) without GU wobbles (C) and with at most one GU wobble (D).