Because previously used high-throughput methods were unable to determine the amount of protein repression, the relative contributions of mRNA destabilization and translational repression during miRNA-mediated regulation has been of intense interest. Our miR-223 data was informative for addressing this issue because it examined the response, at both the mRNA and the protein level, of removing an endogenous miRNA, without the confounding influences of exogenous targeting mediated by an ectopically delivered miRNA. The near steady-state nature of our miR-223 system also avoided quantification caveats inherent to transient transfection, such as variable transfection efficiencies and pre-steady-state complexities especially acute when comparing effects on an mRNA to those on its protein because messages and their proteins can have very different intrinsic stabilities. Note that our mRNA quantification used standard array platforms, which include oligo(dT) priming during detection, and thus the mRNA destabilization we observed encompassed the conversion of the message into a form that was unsuitable for translation because it lacked a poly(A) tail.
To achieve greater quantification accuracy in this analysis of individual proteins, we narrowed our focus to the 2,773 proteins quantified with ≥6 independent measurements. Plotting protein changes as a function of mRNA changes indicated a strong positive correlation for messages with 7mer or 8mer 3′-UTR sites (; r2
= 0.45 and 0.63, P
, respectively) and weaker correlation for messages without sites (; r2
= 0.15, P
, Pearson’s correlation test). Proteins in both plots displayed some scatter around the origin; however, when normalizing to those without sites, many more of those from messages with sites increased in response to miR-223 loss ( and Supplementary Fig. 6
). Immunoblots probing for three of the more responsive proteins confirmed protein derepression in mir-223-/Y
neutrophils differentiated in vitro
as well as in those isolated directly from mice (Supplementary Fig. 7
Comparison of protein and mRNA changes accompanying miR-223 loss
Two of the three most responsive proteins derived from messages with single, non-conserved 7mers ()—sites that on their own would not be expected to impart such a robust response. Previous work has shown that sites falling within 8-40 nucleotides of sites to co-expressed miRNAs typically act cooperatively, which increases the effect of loosing interactions at particular sites7
. We performed high-throughput sequencing to identify miRNAs co-expressed in cultured neutrophils (Supplementary Table 2
) and found that both of the highly responsive 7mers fell near to sites matching a co-expressed miRNA, with intersite spacing favouring a cooperative response (). The site in Ctsl
was near a site for the miR-26 family, one of five families sequenced more frequently than miR-223, whereas the site in Gns
fell near a site to the miR-103/107 family, sequenced about a third as often as miR-223 (Supplementary Table 2
The most responsive proteins deriving from messages with at least one 7-8mer 3′-UTR site
If protein changes merely reflected mRNA changes, with no additional repression at the translational level, then the points would fall on the diagonal (, grey line). Although many were on the diagonal or very close to it, least-squares linear regression yielded a positive y-intercept (+0.053 and +0.079 for 7mer and 8mer data, respectively). These modest yet statistically significantly positive y-intercept values (P = 0.0002 and P = 0.042, t-test) suggested that a cohort of genes were modestly derepressed at the protein level with little or no change at the mRNA level. The messages of such genes were each good candidates for targets affected only at the translational level, although some might have derived from genes undergoing non-miRNA-mediated transcriptional repression as a compensatory feedback response to the loss of miR-223 targeting.
Despite evidence for some translation-only repression, all proteins derepressed by more than 50% (log2 > 0.58) derived from messages that displayed detectable increases ( and ). Moreover, only five points were more than 0.58 units (log2) above the diagonal (, upper dashed line; , indicated with §). Note that a 33% repression by miR-223 in wild-type neutrophils would correspond to a 50% (+0.58 log2) derepression in mutant neutrophils. Thus, in wild-type neutrophils only 5 of the 305 quantified proteins from messages with 7-8mer 3′-UTR sites appeared to undergo translational repression by more than 33%. We conclude that, although in some instances translational repression produces a substantial amount of endogenous miRNA-mediated repression, this occurred for surprisingly few of the many inferred targets. Substantial translational repression appeared so rarely because targets repressed only at the level of translation were repressed quite modestly (<33%); for targets undergoing more robust repression, the major component of the repression was usually mRNA destabilization (). Further study is required to determine whether those mRNA molecules undergoing miRNA-mediated repression might experience translational repression as a prelude to destabilization, but our results show that mRNA destabilization can explain most of the endogenous miR-223-mediated repression.
Our proteomics data were limited to the confidently quantified proteins, which were expected to be those that were both soluble and more highly expressed in neutrophils. To consider how the expression bias might have influenced our results, we plotted the distributions of mRNAs and quantified proteins as a function of mRNA expression in neutrophils, considering all mRNAs of our non-redundant data set (including those without detectable expression), as well as those with 3′-UTR sites (). The messages with conserved or non-conserved 3′-UTR sites displayed the full range of expression values, with a distribution matching that of messages more generally. As anticipated, more quantified proteins derived from highly expressed messages (). However, the distribution of quantified proteins from messages with sites (conserved or non-conserved) closely matched that of those without sites. Moreover, we found no evidence that the greater representation of proteins from more highly expressed messages underrepresented the impact of miRNAs on protein output; if anything, proteins from more highly expressed messages tended to respond more robustly than did those from lowly expressed messages (). An analysis using Gene Ontology terms32
came to similar conclusions (data not shown). Therefore, although our experiment monitored the impact on only a portion of the neutrophil proteome and thus missed many miR-223 targets (including some conserved targets, such as Mef2c; refs 2, 21
), we found no reason to suspect that undetected targets respond more robustly.
The proteins from the least abundant mRNAs appeared to respond without detectable mRNA changes (, ≤6.5 bin). Apparent dominance of the translational component might have been a consequence of less reliable array signals for these messages, many of which fell within background signals from non-expressed messages. A more intriguing possibility is that very efficient translation of these messages (inferred from the ability to quantify proteins from such lowly expressed messages) makes them more susceptible to greater translational repression.