Translational control by ribosome pausing in bacteria: How a non-uniform pace of translation affects protein production and folding

Protein homeostasis of bacterial cells is maintained by coordinated processes of protein production, folding, and degradation. Translational efficiency of a given mRNA depends on how often the ribosomes initiate synthesis of a new polypeptide and how quickly they read the coding sequence to produce a full-length protein. The pace of ribosomes along the mRNA is not uniform: periods of rapid synthesis are separated by pauses. Here, we summarize recent evidence on how ribosome pausing affects translational efficiency and protein folding. We discuss the factors that slow down translation elongation and affect the quality of the newly synthesized protein. Ribosome pausing emerges as important factor contributing to the regulatory programs that ensure the quality of the proteome and integrate the cellular and environmental cues into regulatory circuits of the cell

Translational Regulation in Arabidopsis thaliana: Genetic and Functional Characterization of Eukaryotic Initiation Factor 3

Molecular functions of eukaryotic initiation factor 3 (eIF3) in translation are manifold, encompassing events from initiation complex assembly to translation termination. The contribution of the individual subunits of eIF3 to its multiple activities is quite unclear. It has been hypothesized that several of its 13 subunits contribute to mRNA specific regulation. Prior research had established that the h subunit of eIF3 in Arabidopsis was required for translation of specific mRNAs as well as for organ formation and meristem development. This study aims towards understanding the functions of individual subunits of eIF3 in the context of plant development and to further define the role of eIF3h at the molecular level. This dissertation describes an effort to identify mutations affecting each of the 13 eIF3 subunits. Using a panel of pollen-specific fluorescent marker genes, eIF3 subunits e, h and i1 were demonstrated to be essential for normal male gametophyte development. Furthermore, subunits b and c proved to be essential for embryo development. In contrast, a mutation in eIF3k revealed no phenotypic abnormalities. This work represents a systematic effort to attribute functions to many of the eIF3 subunits in growth and development in a multicellular eukaryote. The h subunit of eIF3 is necessary for the efficient translation of specific mRNAs in Arabidopsis. In particular, eIF3h fosters the translation of those mRNAs that harbor multiple upstream open reading frames (uORFs) in their 5’ leader. The specific molecular activity of eIF3h was investigated by structure-function analysis of the 5\u27 leader of the Arabidopsis AtbZip11 mRNA, which harbors a set of four uORFs that is evolutionarily conserved. By pairing extensive mutagenesis of the AtbZip11 5\u27 leader with gene expression analysis in Arabidopsis seedlings, it was revealed that eIF3h helps the ribosome to retain its reinitiation competence during uORF translation. These data establish a function for the h subunit of eIF3 in a special case of translation initiation, reinitiation. Finally, the molecular events during translation reinitiation were investigated further for a functional cooperation between eIF3h and the large subunit of the ribosome, given that the large ribosomal subunit had been implicated in reinitiation in other biological contexts. Reinitiation profiling using the AtbZip11 leader demonstrated that a protein of the large ribosomal subunit, RPL24B, bolsters reinitiation similar to eIF3h. Taken together, there exists a functional cooperation between the large ribosomal subunit and eIF3 that helps ribosomes to reinitiate after translation of uORFs

The Evolutionary and Functional Roles of Synonymous Codon Usage in Eukaryotes

Most amino acids are encoded by multiple synonymous codons. Although alternative usage of synonymous codons does not affect the amino acid sequences of proteins, researchers have been reporting evidence for functional synonymous codon usage at the species- and gene-specific levels for over four decades. It has been shown that variations in synonymous codon usage can affect phenotypes through diverse mechanisms such as shaping translation efficiency and mRNA stability. On the other hand, the common view that cellular and organismal phenotypes are primarily determined by proteins whose functions are primarily determined by amino acid sequences, often drives the assumption that synonymous mutations are evolutionarily neutral. Consequently, this assumption has been used extensively in evolutionary biology, population genetics, and structural biology. One explanation of the apparent contradiction between the empirical findings, which indicate that synonymous mutations can affect related phenotypes, and the theoretical models, which stipulate that synonymous mutations are neutral, is that neutral synonymous mutations represent the general rule while non-neutral synonymous mutations represent the rare exceptions. In my thesis, I examined this explanation by applying computational and experimental approaches, which indicated that: 1) Non-neutral synonymous mutations significantly affect a considerable proportion of protein-coding genes; 2) Gene-specific codon usage patterns, such as the preference for a specific combination of rare codons, are possibly associated with specific gene functions, such as enhancing tissue-specific gene expression; 3) Some protein-coding genes include codon clusters whose codon usage patterns cannot be explained by selection-independent processes, and thus such codon clusters seem to serve as domains affecting protein functions. Together, these data suggest that synonymous mutations should not be a priori considered neutral. Furthermore, my studies suggest that the biochemical functions of at least some proteins are not only shaped by the constituent amino acid residues but also by codon usage biases at the gene-specific and sub-genic levels. In conclusion, my thesis work suggests that many of the commonly used approaches for analyzing the selection on protein-coding DNA sequences, which rely on the assumption that synonymous mutations are generally neutral, may generate biased results. Furthermore, my studies indicate that selection on gene-specific codon usage bias has evolved to serve diverse biological functions, which are still mostly uncharacterized

Mammalian Cis-Acting RNA Sequence Elements

Cis-acting regulatory sequence elements are sequences contained in the 3′ and 5′ untranslated region, introns, or coding regions of precursor RNAs and mature mRNAs that are selectively recognized by a complementary set of one or more trans-acting factors to regulate posttranscriptional gene expression. This chapter focuses on mammalian cis-acting regulatory elements that had been recently discovered in different regions: pre-processed and mature. The chapter begins with an overview of two large networks of mRNAs that contain conserved AU-rich elements (AREs) or GU-rich elements (GREs), and their role in mammalian cell physiology. Other, less conserved, cis-acting elements and their functional role in different steps of RNA maturation and metabolism will be discussed. The molecular characteristics of pathological cis-acting sequences that rose from gene mutations or transcriptional aberrations are briefly outlined, with the proposed approach to restore normal gene expression. Concise models of the function of posttranscriptional regulatory networks within different cellular compartments conclude this chapter

Transcriptional and translational dynamics of the human heart

Die Genexpression wurde bisher hauptsächlich auf Transkriptions- und Proteinebene untersucht, wobei der Einfluss der Translation, die die Proteinhäufigkeit direkt beeinflusst, weitgehend außer Acht gelassen wurde. Um diese Rolle besser zu verstehen, habe ich Ribosomen-Profiling-Daten (Ribo-seq) verwendet, um die Translationsregulation zu untersuchen und neue Translationsvorgänge in 65 linksventrikulären Proben von DCM-Patienten im Endstadium und 15 Nicht-DCM-Kontrollen zu identifizieren. Dieser Datensatz half dabei, die Transkriptions- und Translationsregulation zwischen erkrankten und nicht betroffenen menschlichen Herzen zu sezieren und enthüllte Gene und Prozesse, die rein unter Translationskontrolle stehen. Darüber hinaus habe ich neue kardiale Proteine vorhergesagt, die von langen nicht-kodierenden RNAs (lncRNAs) und zirkulären RNAs (circRNAs) translatiert werden. Computergestützte Analysen dieser evolutionär jungen Proteine legten eine Beteiligung an verschiedenen molekularen Prozessen nahe, mit einer besonderen Anreicherung für den mitochondrialen Energiestoffwechsel. Schließlich identifizierte ich RNA-bindende Proteine (RBPs), deren Expression die Menge der Ziel-mRNA oder die Frequenz der Translationseffizienz (TE) beeinflusst. Für eine Untergruppe von 21 RBPs habe ich die Regulation auf beiden quantitativen Merkmalen beobachtet, was zu einer unterschiedlichen mechanistischen Basis der Expressionskontrolle für unabhängige Gensätze führte. Obwohl die genaue Umschaltung der RBP-Funktion wahrscheinlich durch eine Kombination von mehreren Faktoren erreicht wird, haben wir für drei Kandidaten eine starke Abhängigkeit von der Zielgenlänge und der 5'-UTR-Struktur beobachtet. Diese Arbeit präsentiert einen Katalog von neu identifizierten Translationsereignissen und einen quantitativen Ansatz zur Untersuchung der Translationsregulation im gesunden und kranken menschlichen Herzen.Gene expression has primarily been studied on transcriptional and protein levels, largely disregarding the extent of translational regulation that directly influences protein abundance. To elucidate its role, I used ribosome profiling (Ribo-seq) data, obtained through ribosome profiling, to study translational regulation and identify novel translation events in 65 left ventricular samples of end-stage DCM patients and 15 non-DCM controls. This dataset helped dissect transcriptional and translational regulation between diseased and unaffected human hearts, revealing genes and processes purely under translational control. These would have remained undetected by only looking at the transcriptional level. Furthermore, I predicted novel cardiac proteins translated from long non-coding RNAs (lncRNAs) and circRNAs. Computational analysis of these evolutionary young proteins suggested involvement in diverse molecular processes with a particular enrichment for mitochondrial processes. Finally, I identified RNA-binding proteins (RBPs) whose expression influences target mRNA abundance or translational efficiency (TE) rates. For a subset of 21 RBPs, I have observed regulation on both quantitative traits, which resulted in different mechanistic basis expression control for independent sets of genes. Though the precise switch in RBP function is likely achieved by a combination of multiple factors, for three candidates we have observed a strong dependency on target length and 5’ UTR structure. This work presents a catalogue of newly identified translation events and a quantitative approach to study translational regulation in the healthy and failing human heart

Analysis of translation of 5’ untranslated regions in cancer

Short upstream open reading frames (uORFs) are cis-acting elements located within the 5'-leader sequence of transcripts. Recent genome-wide ribosome profiling (RiboSeq) studies have demonstrated the widespread presence of uORFs in the transcriptome and have shown that many uORFs can initiate with non-AUG codons. uORFs can impact gene expression of the downstream main open reading frame (mORF) by triggering messenger RNA (mRNA) decay or by regulating translation. Thus, disruption, elimination or creation of uORFs can elicit the development of several genetic diseases, such as cancer. The ATP-binding cassette subfamily E member 1 (ABCE1) gene belongs to the ABC gene transporter superfamily. However, it does not behave as a drug transporter like the other members of this family. ABCE1 actively participates in the different stages of the translation process and is involved in cell proliferation and anti-apoptotic signaling processes, associating ABCE1 to a potential oncogenic function. RiboSeq occupancy profiles of the ABCE1 mRNA 5’-leader sequence indicate an active translation associated with the presence of uORFs, which is suggestive of a high translational regulation. Our aim was to study the translational regulation mediated by the five AUG and five non-AUG uORFs present in the human ABCE1 5’-leader sequence in colorectal cancer. With this purpose, we constructed a set of Firefly luciferase (FLuc) reporter vectors derived from the wild-type one containing the native configuration of the human ABCE1 5’-leader sequence upstream of the FLuc ORF, and transiently transfected colorectal cancer HCT116 cells. Here we show that ABCE1 mORF expression is regulated by its uORFs. Our results are consistent with a model wherein uORF1 recruits ribosomes onto the mRNA, behaving like a ribosomal barrier. The ribosomes that efficiently bypass uORF1 and/or uORF2, must probably reinitiate at uORF3 and/or uORF5, while uORF4 is greatly bypassed. uORF3 and uORF5 function as repressive uORFs that may cooperate to reach a maximum repression of the mORF. Thus, both bypass and reinitiation events of the AUG uORFs within ABCE1 5’-leader sequence contribute for the translational control of the mORF. In constrast, the non-AUG uORFs seem to be devoid of a significant inhibitory activity. The AUG uORF-mediated translational control is maintained in normal and in endoplasmic reticulum (ER) stress conditions, which keeps the expression level of ABCE1 at a minimum, showing that ABCE1 is a stress-resistant transcript whose functions are equally essential in normal and in coditions of global translation impairment. In addition, we show that ABCE1 uORF-mediated translational regulation is preserved in non-tumorigenic and cancerous cells, which is consistent with a lack of an oncogenic function by the uORFs, as well as ABCE1 itself, in the colorectal cancer cell line tested. This study contributes with an additional example of how uORF-mediated translational regulation can occur. In addition, it reveals how important is to screen the 5’-leader sequence of the transcripts in search for potential disease-related variants. This information might be relevant for the implementation of new diagnostic and/or therapeutic tools for diseases associated with the deregulation of uORF-mediated translational control.BioISI – Biosystems & Integrative Sciences Institute da Faculdade de Ciências da Universidade de Lisboa (UID/MULTI/04046/2013

Biosynthesis of pyranonaphthoquinone polyketides:characterization of the alnumycin pathway

Alnumycin A is an aromatic pyranonaphthoquinone (PNQ) polyketide closely related to the model compound actinorhodin. While some PNQ polyketides are glycosylated, alnumycin A contains a unique sugar-like dioxane moiety. This unusual structural feature made alnumycin A an interesting research target, since no information was available about its biosynthesis. Thus, the main objective of the thesis work became to identify the steps and the enzymes responsible for the biosynthesis of the dioxane moiety. Cloning, sequencing and heterologous expression of the complete alnumycin gene cluster from Streptomyces sp. CM020 enabled the inactivation of several alnumycin biosynthetic genes and preliminary identification of the gene products responsible for pyran ring formation, quinone formation and dioxane biosynthesis. The individual deletions of the genes resulted in the production of several novel metabolites, which in many cases turned out to be pathway intermediates and could be used for stepwise enzymatic reconstruction of the complete dioxane biosynthetic pathway in vitro. Furthermore, the in vitro reactions with purified alnumycin biosynthetic enzymes resulted in the production of other novel compounds, both pathway intermediates and side products. Identification and molecular level studies of the enzymes AlnA and AlnB catalyzing the first step of dioxane biosynthesis – an unusual C-ribosylation step – led to a mechanistic proposal for the C-ribosylation of the polyketide aglycone. The next step on the dioxane biosynthetic pathway was found to be the oxidative conversion of the attached ribose into a highly unusual dioxolane unit by Aln6 belonging to an uncharacterized protein family, which unexpectedly occurred without any apparent cofactors. Finally, the last step of the pathway was found to be catalyzed by the NADPH-dependent reductase Aln4, which is able to catalyze the conversion of the formed dioxolane into a dioxane moiety. The work presented here and the knowledge gained of the enzymes involved in dioxane biosynthesis enables their use in the rational design of novel compounds containing C–C bound ribose, dioxolane and dioxane moieties.Siirretty Doriast

Biochemical analysis of translational recording driven by 2A peptide

PhD2A/2A-like peptides are short sequences (20-30 amino acids) encoded predominantly within open reading frames (ORFs) of RNA viruses. They drive a non-canonical translation, in which the nascent chain is released from the ribosome at a sense (proline) codon, followed by continued translation to generate a separate downstream protein, initiated from the same proline codon. The aim of this study is to investigate the role of ribosomal factors in the 2A reaction in Saccharomyces cerevisiae cells. Results obtained showed that reduced activity of eRF1/3 inhibits the 2A reaction. This inhibition did not strongly correlate with the effect that mutations have on termination at stop codons. In particular, several mutations within the NIKS motif, which is essential for stop codon recognition, had minimal effect on the 2A reaction. To confirm these results, we developed a new reporter to investigate the 2A activity, where the green fluorescent protein (GFP) sequence was separated with a 2A sequence, between residues 157 and 158. This reporter was utilised to confirm the effects of eRF1 mutations, previously assessed by immunoprecipitation, and results, observed by flow cytometry, revealed consistency in terms of the role of eRFs in the 2A reaction. In summary, these observations provide evidences supporting recruitment of eRFs to the ribosome to drive the non-canonical termination event that releases the first part of the 2A reaction.The Ministry of Higher Education and the University of Mosul/IRA