mRNA 5' terminal sequences drive 200-fold differences in expression through effects on synthesis, translation and decay.

mRNA regulatory sequences control gene expression at multiple levels including translation initiation and mRNA decay. The 5' terminal sequences of mRNAs have unique regulatory potential because of their proximity to key post-transcriptional regulators. Here we have systematically probed the function of 5' terminal sequences in gene expression in human cells. Using a library of reporter mRNAs initiating with all possible 7-mer sequences at their 5' ends, we find an unexpected impact on transcription that underlies 200-fold differences in mRNA expression. Library sequences that promote high levels of transcription mirrored those found in native mRNAs and define two basic classes with similarities to classic Initiator (Inr) and TCT core promoter motifs. By comparing transcription, translation and decay rates, we identify sequences that are optimized for both efficient transcription and growth-regulated translation and stability, including variants of terminal oligopyrimidine (TOP) motifs. We further show that 5' sequences of endogenous mRNAs are enriched for multi-functional TCT/TOP hybrid sequences. Together, our results reveal how 5' sequences define two general classes of mRNAs with distinct growth-responsive profiles of expression across synthesis, translation and decay.

Measuring mRNA Decay with Roadblock-qPCR.

The control of mRNA stability is fundamental to gene regulation, and a deeper understanding of this post-transcriptional regulatory step can provide key insights into gene function. Measuring mRNA half-lives directly, however, is challenging. The most common strategies for evaluating mRNA stability and decay involve blocking general transcription and then measuring the decline in mRNA levels over time. The downside of these approaches, however, is that they severely impact cell function and viability, indirectly perturbing gene expression. Here, we describe Roadblock-qPCR, a simple method for measuring mRNA decay kinetics in living cells that is both economical and quick. Cells are first incubated with the nucleoside analog 4-thiouridine (4sU), which is readily incorporated into nascent mRNAs during transcription. RNA is then extracted and treated with N-ethylmaleimide (NEM), a sulfhydryl alkylating agent that selectively modifies 4sU, before proceeding to cDNA synthesis. Because the NEM-modified 4sU creates a chemical "roadblock" that interferes with reverse transcription, this treatment ultimately results in the depletion of the nascent 4sU-containing transcripts from the cDNA pool. As such, the decay rate of the non-4sU-labeled pre-existing mRNAs can be monitored by quantitative PCR (qPCR). In combination with spike-in standards, this approach can be used to efficiently and accurately measure the half-lives of endogenous mRNAs with a wide range of stabilities, while avoiding the artifacts of transcription shutoff strategies. © 2022 Wiley Periodicals LLC. Basic Protocol: Roadblock-qPCR Support Protocol: Synthesis of spike-in mRNA.

Global analysis of LARP1 translation targets reveals tunable and dynamic features of 5' TOP motifs.

Terminal oligopyrimidine (TOP) motifs are sequences at the 5' ends of mRNAs that link their translation to the mTOR Complex 1 (mTORC1) nutrient-sensing signaling pathway. They are commonly regarded as discrete elements that reside on ∼100 mRNAs that mostly encode translation factors. However, the full spectrum of TOP sequences and their prevalence throughout the transcriptome remain unclear, primarily because of uncertainty over the mechanism that detects them. Here, we globally analyzed translation targets of La-related protein 1 (LARP1), an RNA-binding protein and mTORC1 effector that has been shown to repress TOP mRNA translation in a few specific cases. We establish that LARP1 is the primary translation regulator of mRNAs with classical TOP motifs genome-wide, and also that these motifs are extreme instances of a broader continuum of regulatory sequences. We identify the features of TOP sequences that determine their potency and quantify these as a metric that accurately predicts mTORC1/LARP1 regulation called a TOPscore. Analysis of TOPscores across the transcriptomes of 16 mammalian tissues defines a constitutive "core" set of TOP mRNAs, but also identifies tissue-specific TOP mRNAs produced via alternative transcription initiation sites. These results establish the central role of LARP1 in TOP mRNA regulation on a transcriptome scale and show how it connects mTORC1 to a tunable and dynamic program of gene expression that is tailored to specific biological contexts.