Kristian E. Baker

Codon optimality is a major determinant of mRNA stability

mRNA degradation represents a critical regulated step in gene expression. Although the major pathways in turnover have been identified, accounting for disparate half-lives has been elusive. We show that codon optimality is one feature that contributes greatly to mRNA stability. Genome-wide RNA decay analysis revealed that stable mRNAs are enriched in codons designated optimal, whereas unstable mRNAs contain predominately non-optimal codons. Substitution of optimal codons with synonymous, non-optimal codons results in dramatic mRNA destabilization, whereas the converse substitution significantly increases stability. Further, we demonstrate that codon optimality impacts ribosome translocation, connecting the processes of translation elongation and decay through codon optimality. Finally, we show that optimal codon content accounts for the similar stabilities observed in mRNAs encoding proteins with coordinated physiological function. This work demonstrates that codon optimization exists as a mechanism to finely tune levels of mRNAs and, ultimately, proteins.

Co-translational mRNA decay in Saccharomyces cerevisiae

The rates of RNA decay and transcription determine the steady-state levels of all messenger RNA and both can be subject to regulation. Although the details of transcriptional regulation are becoming increasingly understood, the mechanism(s) controlling mRNA decay remain unclear. In yeast, a major pathway of mRNA decay begins with deadenylation followed by decapping and 5′–3′ exonuclease digestion. Importantly, it is hypothesized that ribosomes must be removed from mRNA before transcripts are destroyed. Contrary to this prediction, here we show that decay takes place while mRNAs are associated with actively translating ribosomes. The data indicate that dissociation of ribosomes from mRNA is not a prerequisite for decay and we suggest that the 5′–3′ polarity of mRNA degradation has evolved to ensure that the last translocating ribosome can complete translation.

Decapping of long noncoding RNAs regulates inducible genes.

Decapping represents a critical control point in regulating expression of protein coding genes. Here, we demonstrate that decapping also modulates expression of long noncoding RNAs (lncRNAs). Specifically, levels of >100 lncRNAs in yeast are controlled by decapping and are degraded by a pathway that occurs independent of decapping regulators. We find many lncRNAs degraded by DCP2 are expressed proximal to inducible genes. Of these, we show several genes required for galactose utilization are associated with lncRNAs that have expression patterns inversely correlated with their mRNA counterpart. Moreover, decapping of these lncRNAs is critical for rapid and robust induction of GAL gene expression. Failure to destabilize a lncRNA known to exert repressive histone modifications results in perpetuation of a repressive chromatin state that contributes to reduced plasticity of gene activation. We propose that decapping and lncRNA degradation serve a vital role in transcriptional regulation specifically at inducible genes.

Ectopic RNase E sites promote bypass of 5′‐end‐dependent mRNA decay in Escherichia coli

In Escherichia coli, 5′‐terminal stem–loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear. A synthetic 5′‐terminal hairpin stabilizes the rpsT mRNA sixfold. This stabilization is dependent on efficient translational initiation and ribosome transit through at least two‐thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA. Insertion of a 12–15 residue ‘ectopic’ RNase E cleavage site from either the rne leader or 9S pre‐rRNA into the 5′‐non‐coding region of the rpsT mRNA significantly reduces the stabilizing effect of the terminal stem–loop, dependent on RNase E. A similar insertion into the rpsT coding sequence is partially destabilizing. These findings demonstrate that RNase E can bypass an interaction with the 5′‐terminus, and exploit an alternative ‘internal entry’ pathway. We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5′‐termini, the use of internal entry for bypass of barriers to decay, ‘ectopic sites’ and the role of translating ribosomes.

Translation of Small Open Reading Frames within Unannotated RNA Transcripts in Saccharomyces cerevisiae

High-throughput gene expression analysis has revealed a plethora of previously undetected transcripts in eukaryotic cells. In this study, we investigate >1,100 unannotated transcripts in yeast predicted to lack protein-coding capacity. We show that a majority of these RNAs are enriched on polyribosomes akin to mRNAs. Ribosome profiling demonstrates that many bind translocating ribosomes within predicted open reading frames 10-96 codons in size. We validate expression of peptides encoded within a subset of these RNAs and provide evidence for conservation among yeast species. Consistent with their translation, many of these transcripts are targeted for degradation by the translation-dependent nonsense-mediated RNA decay (NMD) pathway. We identify lncRNAs that are also sensitive to NMD, indicating that translation of noncoding transcripts also occurs in mammals. These data demonstrate transcripts considered to lack coding potential are bona fide protein coding and expand the proteome of yeast and possibly other eukaryotes.

Nonsense-mediated mRNA decapping occurs on polyribosomes in Saccharomyces cerevisiae

Nonsense-mediated decay (NMD) degrades mRNA containing premature translation termination codons. In yeast, NMD substrates are decapped and digested exonucleolytically from the 5′ end. Despite the requirement for translation in recognition, degradation of nonsense-containing mRNA is considered to occur in ribosome-free cytoplasmic P bodies. We show decapped nonsense-containing mRNA associate with polyribosomes, indicating that recognition and degradation are tightly coupled and that polyribosomes are major sites for degradation of aberrant mRNAs.

Nonsense-mediated RNA decay - a switch and dial for regulating gene expression

Nonsense‐mediated RNA decay (NMD) represents an established quality control checkpoint for gene expression that protects cells from consequences of gene mutations and errors during RNA biogenesis that lead to premature termination during translation. Characterization of NMD‐sensitive transcriptomes has revealed, however, that NMD targets not only aberrant transcripts but also a broad array of mRNA isoforms expressed from many endogenous genes. NMD is thus emerging as a master regulator that drives both fine and coarse adjustments in steady‐state RNA levels in the cell. Importantly, while NMD activity is subject to autoregulation as a means to maintain homeostasis, modulation of the pathway by external cues provides a means to reprogram gene expression and drive important biological processes. Finally, the unanticipated observation that transcripts predicted to lack protein‐coding capacity are also sensitive to this translation‐dependent surveillance mechanism implicates NMD in regulating RNA function in new and diverse ways.

Analysis of Nonsense‐Mediated mRNA Decay in Saccharomyces cerevisiae

Nonsense‐mediated mRNA decay is a highly conserved pathway that degrades mRNAs with premature termination codons. These mRNAs include mRNAs transcribed from nonsense or frameshift alleles as well as wild‐type mRNA with signals that direct ribosomes to terminate prematurely. This unit describes techniques to monitor steady‐state mRNA levels, decay rates, and structural features of mRNAs targeted by this pathway, as well as in vivo analysis of nonsense suppression and allosuppression in the yeast Saccharomyces cerevisiae. Protocols for the structural features of mRNA include analysis of cap status, 5′ and 3′ untranslated region (UTR) lengths, and poly(A) tail length. Curr. Protoc. Cell Biol. 54:27.3.1‐27.3.39.

Purification of Transcript-Specific mRNP Complexes Formed In Vivo from Saccharomyces cerevisiae

RNA binding proteins play critical roles in shaping the complex life cycle of cellular transcripts. For most RNAs, the association with a distinct complement of proteins serves to orchestrate its unique pattern of maturation, localization, translation, and stability. A key aspect to understanding how transcripts are differentially regulated lies, therefore, in the ability to identify the particular repertoire of protein binding partners associated with an individual transcript. We describe here an optimized experimental procedure for purifying a single mRNA population from yeast cells for the characterization of transcript-specific mRNA-protein complexes (mRNPs) as they exist in vivo. Chemical cross-linking is used to trap native mRNPs and facilitate the co-purification of protein complexes associated with an individual transcript population that is captured under stringent conditions from cell lysates through hybridization to complementary DNA oligonucleotides. The resulting mRNP is highly enriched and largely devoid of non-target transcripts, and can be used for a number of downstream analyses including protein identification by mass spectrometry.

The Centrality of RNA, Then and Now

Today, the critical roles of RNA in information transfer are taken for granted. Most graduate students can recite the basic rubrics of molecular biology; DNA is transcribed to make pre-mRNA, which is then extensively processed in the nucleus to yield mRNA. The mRNA is then recruited to ribosomes, where with the obligatory aid of decoding tRNA, it is translated 5′ to 3′ to make proteins, amino to carboxyl. Remarkably, peptide bond formation is catalyzed by ribosomal RNA itself.View Large Image | View Hi-Res Image | Download PowerPoint SlideAlthough these “facts” are well known, it is only the very rare student or even faculty member who can describe how we came to learn these fundamentals. Ask how mRNA was discovered, how the genetic code was deciphered, or how the directionality of protein synthesis was established, and one is more than likely to encounter blank stares. Fortunately, there is now a straightforward and enjoyable way to transform those stares into cogent answers by reading James Darnells book on the history of RNA research.Darnell tells the story of the early days of the molecular biology of RNA through the eyes of a prolific scientist who lived through those exciting times while making seminal contributions himself. The narrative begins in an era when the concept of macromolecules of any kind was met with stubborn and today unthinkable resistance. Nevertheless, these misguided ideas were dispelled in a series of breakthroughs such as the crystallization of urease and pepsin, the sequencing of insulin, and the elucidation of secondary structural elements in proteins such as the alpha helix. The early history of genetics in peas, the fly, and bacteria is also described, as are the famous experiments that showed quite convincingly that DNA was the genetic material.After the elucidation of the double-helical structure of DNA, researchers turned toward trying to understand how the information in DNA was interpreted to make protein. Darnell takes the reader down the paths that lead to the discovery of mRNA. Looking back, it is remarkable how much was leaned from “simple” approaches such as base composition analysis of labeled RNA (with [32P] orthophosphate) and gradient ultracentrifugation, techniques that provided the first hints of mRNA in bacteriophage-infected bacteria. These experiments presaged an avalanche of studies that showed among other things that ribosomes are the sites of protein synthesis and demonstrated the existence of transfer RNAs (then called soluble RNAs). However, the most important work of the time was the analysis by Jacob and Monod of lactose metabolism in bacteria. Their discovery of the lac operon and elucidation of its regulatory circuitry were profoundly influential at the time and still drive our perceptions of gene control today. Darnell describes their work in some detail, but unfortunately, some overzealous (or underzealous) proofreader changed the famous PaJaMo (Pardee, Jacob, Monod) experiment to the PaJaMa experiment, perhaps to bestow a catchier moniker.The most striking aspect of this period of intense discovery is that the field was driven by the work of a remarkably small group of investigators dominated by the genius of Jacob, Monod, Crick, and Brenner. Moreover, in the days before e-mail and FedEx, the level of communication between the groups involved appears to be amazingly efficient. The confluence of shared ideas and productive collaborations certainly drove the field forward. It makes one long for those “simpler” days when there were not thousands of journals to source and hundreds of meetings to attend where only published work is presented.Following the discovery of mRNA, interest turned toward deciphering the genetic code. Darnell lucidly takes the reader through the thinking at the time and describes the famous “n = 3 experiment,” a clever genetic analysis proving that nonoverlapping triplets of nucleotides were the code words. He then details how Ochoa and Nirenberg broke the code, each using different experimental approaches. With the solving of this fundamental problem the book shifts gears and departs the prokaryotic world to enter the maze of eukaryotic RNA metabolism. It is worth noting that the story up to this point closely parallels the content of another engaging book, The Eighth Day of Creation by Horace Freeland Judson, an eminently readable and highly detailed account of the early days of molecular biology told from the perspective of an historian of science but not a scientist himself.As Darnell delves into eukaryotic RNA metabolism, the tone of the book changes and becomes more personal, undoubtedly because Darnell himself was intimately involved in this area of research. He recounts the development and importance of tissue culture cells and animal and plant viral propagation without which the work would have been impossible. The reader is led through the discovery of ribosomal RNA processing and tRNA processing and learns that it was Darnell himself that coined the term “RNA processing.” Nevertheless, the most engaging part of this portion of the book is the description of the mystery of heterogenous (in length) nuclear RNA, hnRNA. Pulse labeling of HeLa cells revealed the presence of hnRNA, and base composition analysis showed that it was distinct from ribosomal RNA. Subsequent to the discovery of the 5′ methylated cap and serendipitous discovery of 3′ polyadenylate tails (interesting stories in their own right), it was shown that hnRNA possessed both. However, much of the label in hnRNA never left the nucleus. Although labeling strategies suggested a precursor product relationship between hnRNA and mRNA, it remained unclear how hnRNA was reduced in size. A number of clever and technically difficult approaches such as UV transcription mapping successively narrowed down the possibilities, and Darnell describes how tantalizingly close his group was to discovering splicing. However, this was not to be as the Roberts and Sharp groups proved using electron microscopy that removal of intervening sequences (introns) occurred in viral pre-mRNAs. Soon after, it became clear that splicing caused the reduction in size of hnRNA to mRNA. Following the discovery of splicing, the (at the time) astounding discovery of catalytic RNA is briefly described, and the attention is turned toward transcription and its regulation in animal cells.To RNA biologists, the detailed account of the discovery of eukaryotic RNA polymerases and the subsequent description of transcription factors and their roles in controlling gene expression seem like a distraction because, perhaps unfortunately, transcription and RNA processing have evolved into quite distinct fields. The overwhelming complexity of each precludes an in-depth discussion of them both. Nevertheless, the large portion of the book devoted to transcription as well as chromatin and epigenetic histone marks serves as an excellent introduction to these topics for students and faculty alike. One minor criticism is that transcription and its control could be seen to be discussed at the expense of other interesting topics. For example, eukaryotic mRNA turnover is presented in a little over a page, and mRNA translational control is not even mentioned. This aside, to his credit Darnell does cover nearly every aspect of modern RNA biology, and the referencing is remarkably current. Although one may have preferred more detailed treatments of small noncoding RNAs such as microRNA, piwi-interacting RNAs, and small-interfering RNAs, the references provide ready access to anyone who wants to explore these topics in more depth.The final chapter of the book provides a nice summary of the RNA World hypothesis. In a nutshell, a number of lines of evidence suggest that RNA was “the” molecule (because of its catalytic and informational capacities) essential for the origin of life. Perhaps this explains the title of the book, which is to say that RNA was indispensible for life, as we know it, to have begun. In this view, RNase P, the splicesome, the ribosome, and telomerase, among others, are relics of an ancestral “life form” that preceded proteins and DNA. It is clear that the RNA World hypothesis will continue to stimulate new ideas and experimental analyses.In sum, Darnell has succeeded in writing an appealing and cogent account of the rise of RNA molecular biology and its continued centrality in research today. This is an excellent book that should be required reading for graduate students and more senior investigators alike. The emphasis on hypotheses-driven experimental analysis and inclusion of informative figures, many showing primary data, are a significant plus as is the up-to-date referencing. Although quite technical in parts, the concepts are explained in clear enough terms that any reader of Cell should be able to understand and appreciate them.