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The maternal-to-zygotic transition (MZT) is a conserved developmental process where the maternally-derived protein and mRNA cache is replaced with newly made zygotic gene products. We have previously shown that in Drosophila the deposited RNA-binding proteins ME31B, Cup, and Trailer Hitch (TRAL) are ubiquitylated by the CTLH E3 ligase and cleared. However, the organization and regulation of the CTLH complex remain poorly understood in flies. In particular, Drosophila lacks an identifiable substrate adaptor, and the mechanisms restricting degradation of ME31B and its cofactors to the MZT are unknown. Here, we show that the developmental specificity of the CTLH complex is mediated by multipronged regulation, including transcriptional control by the transcription factor OVO and autoinhibition of the E3 ligase. One major regulatory target is the subunit Muskelin, which we demonstrate acts as a substrate adaptor for the Drosophila CTLH complex. Although conserved, Muskelin has structural roles in other species, suggesting a surprising functional plasticity. Finally, we find that Muskelin has few targets beyond the three known RNA binding proteins, showing exquisite target specificity. Thus, multiple levels of integrated regulation restrict the activity of the embryonic CTLH complex to early embryogenesis, seemingly with the goal of regulating three important RNA binding proteins.
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Giardia lamblia is a deeply branching protist and a human pathogen. Its unusual biology presents the opportunity to explore conserved and fundamental molecular mechanisms. We determined the structure of the G. lamblia 80S ribosome bound to tRNA, mRNA, and the antibiotic emetine by cryo-electron microscopy, to an overall resolution of 2.49 Å. The structure reveals rapidly evolving protein and nucleotide regions, differences in the peptide exit tunnel, and likely altered ribosome quality control pathways. Examination of translation initiation factor binding sites suggests these interactions are conserved despite a divergent initiation mechanism. Highlighting the potential of G. lamblia to resolve conserved biological principles; our structure reveals the interactions of the translation inhibitor emetine with the ribosome and mRNA, thus providing insight into the mechanism of action for this widely used antibiotic. Our work defines key questions in G. lamblia and motivates future experiments to explore the diversity of eukaryotic gene regulation.
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Giardia lamblia , Humanos , Giardia lamblia/genética , Giardia lamblia/química , Giardia lamblia/metabolismo , Emetina/farmacología , Emetina/análisis , Emetina/metabolismo , Microscopía por Crioelectrón , Ribosomas/química , ARN Mensajero/metabolismo , AntibacterianosRESUMEN
Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger messenger RNA (mRNA) degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting additional mechanisms by which codon usage regulates gene expression. Using reporters in human and Drosophila cells, we find that transcript levels account for less than half of the variation in protein abundance due to codon usage. This discrepancy is explained by translational differences whereby nonoptimal codons repress translation initiation. Nonoptimal transcripts are also less bound by the translation initiation factors eIF4E and eIF4G1, providing a mechanistic explanation for their reduced initiation rates. Importantly, translational repression can occur without mRNA decay and deadenylation, and it does not depend on the known nonoptimality sensor, CNOT3. Our results reveal a potent mechanism of regulation by codon usage where nonoptimal codons repress further rounds of translation.
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Uso de Codones , Ribosomas , Animales , Humanos , Ribosomas/metabolismo , Biosíntesis de Proteínas , Codón/genética , Codón/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas/metabolismo , Drosophila/genética , Drosophila/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Interview with Olivia Rissland, who studies post-transcriptional gene regulation at the University of Colorado.
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Messenger RNAs (mRNAs) in higher eukaryotes that encode proteins important for the assembly of the translational apparatus (e.g., ribosomal proteins) often harbor a pyrimidine-rich motif at the extreme 5' end known as a 5' terminal oligopyrimidine (5'TOP) sequence. Members of the La-related protein 1 (LARP1) family control 5'TOP expression through a conserved DM15 motif, but the mechanism is not well understood. 5'TOP motifs have not been described in many lower organisms, and fission yeast harbors a LARP1 homolog that also lacks a DM15 motif. In this work, we show that the fission yeast LARP1 homolog, Slr1p, controls the translation and stability of mRNAs encoding proteins analogous to 5'TOP mRNAs in higher eukaryotes, which we thus refer to as proto-5'TOPs. Our data suggest that the LARP1 DM15 motif and the mRNA 5'TOP motif may be features that were scaffolded over a more fundamental mechanism of LARP1-associated control of gene expression.
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Schizosaccharomyces , ARN Mensajero/genética , ARN Mensajero/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Ribosómicas/metabolismo , Biosíntesis de ProteínasRESUMEN
In this issue of Molecular Cell, Gasparski et al.1 and Loedige et al.2 reshape our understanding of subcellular gene product localization by highlighting the importance of messenger RNA (mRNA) stability and co-translational mechanisms in mRNA and protein localization.
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Automóviles , Estabilidad del ARN , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Biosíntesis de ProteínasRESUMEN
Transcriptional changes in Rett syndrome (RTT) are assumed to directly correlate with steady-state mRNA levels, but limited evidence in mice suggests that changes in transcription can be compensated by post-transcriptional regulation. We measure transcription rate and mRNA half-life changes in RTT patient neurons using RATEseq, and re-interpret nuclear and whole-cell RNAseq from Mecp2 mice. Genes are dysregulated by changing transcription rate or half-life and are buffered when both change. We utilized classifier models to predict the direction of transcription rate changes and find that combined frequencies of three dinucleotides are better predictors than CA and CG. MicroRNA and RNA-binding Protein (RBP) motifs are enriched in 3'UTRs of genes with half-life changes. Nuclear RBP motifs are enriched on buffered genes with increased transcription rate. We identify post-transcriptional mechanisms in humans and mice that alter half-life or buffer transcription rate changes when a transcriptional modulator gene is mutated in a neurodevelopmental disorder.
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Síndrome de Rett , Humanos , Ratones , Animales , Síndrome de Rett/genética , ARN Mensajero , Semivida , Proteína 2 de Unión a Metil-CpG/metabolismo , Regulación de la Expresión GénicaRESUMEN
The contribution of mRNA half-life is commonly overlooked when examining changes in mRNA abundance during development. mRNA levels of some genes are regulated by transcription rate only, but others may be regulated by mRNA half-life only shifts. Furthermore, transcriptional buffering is predicted when changes in transcription rates have compensating shifts in mRNA half-life resulting in no change to steady-state levels. Likewise, transcriptional boosting should result when changes in transcription rate are accompanied by amplifying half-life shifts. During neurodevelopment there is widespread 3'UTR lengthening that could be shaped by differential shifts in the stability of existing short or long 3'UTR transcript isoforms. We measured transcription rate and mRNA half-life changes during induced human Pluripotent Stem Cell (iPSC)-derived neuronal development using RATE-seq. During transitions to progenitor and neuron stages, transcriptional buffering occurred in up to 50%, and transcriptional boosting in up to 15%, of genes with changed transcription rates. The remaining changes occurred by transcription rate only or mRNA half-life only shifts. Average mRNA half-life decreased two-fold in neurons relative to iPSCs. Short gene isoforms were more destabilized in neurons and thereby increased the average 3'UTR length. Small RNA sequencing captured an increase in microRNA copy number per cell during neurodevelopment. We propose that mRNA destabilization and 3'UTR lengthening are driven in part by an increase in microRNA load in neurons. Our findings identify mRNA stability mechanisms in human neurodevelopment that regulate gene and isoform level abundance and provide a precedent for similar post-transcriptional regulatory events as other tissues develop.
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The maternal-to-zygotic transition (MZT) is a conserved embryonic process in animals where developmental control shifts from the maternal to zygotic genome. A key step in this transition is zygotic transcription, and deciphering the MZT requires classifying newly transcribed genes. However, due to current technological limitations, this starting point remains a challenge for studying many species. Here, we present an alternative approach that characterizes transcriptome changes based solely on RNA-seq data. By combining intron-mapping reads and transcript-level quantification, we characterized transcriptome dynamics during the Drosophila melanogaster MZT. Our approach provides an accessible platform to investigate transcriptome dynamics that can be applied to the MZT in nonmodel organisms. In addition to classifying zygotically transcribed genes, our analysis revealed that over 300 genes express different maternal and zygotic transcript isoforms due to alternative splicing, polyadenylation, and promoter usage. The vast majority of these zygotic isoforms have the potential to be subject to different regulatory control, and over two-thirds encode different proteins. Thus, our analysis reveals an additional layer of regulation during the MZT, where new zygotic transcripts can generate additional proteome diversity.
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Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Animales , Drosophila melanogaster/metabolismo , Intrones/genética , Cigoto , Transcriptoma/genética , Desarrollo Embrionario/genéticaRESUMEN
Since the initial reported discovery of SARS-CoV-2 in late 2019, genomic surveillance has been an important tool to understand its transmission and evolution. Here, we sought to describe the underlying regional phylodynamics before and during a rapid spreading event that was documented by surveillance protocols of the United States Air Force Academy (USAFA) in late October-November of 2020. We used replicate long-read sequencing on Colorado SARS-CoV-2 genomes collected July through November 2020 at the University of Colorado Anschutz Medical campus in Aurora and the United States Air Force Academy in Colorado Springs. Replicate sequencing allowed rigorous validation of variation and placement in a phylogenetic relatedness network. We focus on describing the phylodynamics of a lineage that likely originated in the local Colorado Springs community and expanded rapidly over the course of two months in an outbreak within the well-controlled environment of the United States Air Force Academy. Divergence estimates from sampling dates indicate that the SARS-CoV-2 lineage associated with this rapid expansion event originated in late October 2020. These results are in agreement with transmission pathways inferred by the United States Air Force Academy, and provide a window into the evolutionary process and transmission dynamics of a potentially dangerous but ultimately contained variant.
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COVID-19 , SARS-CoV-2 , COVID-19/epidemiología , Colorado/epidemiología , Genoma Viral , Humanos , Filogenia , SARS-CoV-2/genéticaRESUMEN
During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3' end formation still appears to be an important regulatory step for gene expression in G. lamblia.
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Giardia lamblia , Poli A , Regiones no Traducidas 3' , Animales , Giardia lamblia/genética , Giardia lamblia/metabolismo , Mamíferos/genética , Poli A/genética , Poli A/metabolismo , Poliadenilación , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
During the COVID-19 pandemic, a shortage of personal protective equipment compromised efficient patient care and provider safety. Volunteers from many different backgrounds worked to meet these demands. Additive manufacturing, laser cutting, and alternative supply chains were used to produce, test, and deliver essential equipment for health care workers and first responders. Distributed equipment included ear guards, face shields, and masks. Contingent designs were created for powered air-purifying respirator hoods, filtered air pumps, intubation shields, and N95 masks.
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COVID-19/epidemiología , Equipos y Suministros/provisión & distribución , Colorado/epidemiología , Diseño de Equipo , Humanos , Máscaras/provisión & distribución , Pandemias , Equipo de Protección Personal/provisión & distribución , SARS-CoV-2 , VoluntariosRESUMEN
The maternal-to-zygotic transition (MZT) is a conserved step in animal development, where control is passed from the maternal to the zygotic genome. Although the MZT is typically considered from its impact on the transcriptome, we previously found that three maternally deposited Drosophila RNA-binding proteins (ME31B, Trailer Hitch [TRAL], and Cup) are also cleared during the MZT by unknown mechanisms. Here, we show that these proteins are degraded by the ubiquitin-proteasome system. Marie Kondo, an E2 conjugating enzyme, and the E3 CTLH ligase are required for the destruction of ME31B, TRAL, and Cup. Structure modeling of the Drosophila CTLH complex suggests that substrate recognition is different than orthologous complexes. Despite occurring hours earlier, egg activation mediates clearance of these proteins through the Pan Gu kinase, which stimulates translation of Kdo mRNA. Clearance of the maternal protein dowry thus appears to be a coordinated, but as-yet underappreciated, aspect of the MZT.
Bestselling author and organizing consultant Marie Kondo has helped people around the world declutter their homes by getting rid of physical items that do not bring them joy. Keeping the crowded environment inside a living cell organized also requires work and involves removing molecules that are no longer needed. A fertilized egg cell, for example, contains molecules from the mother that regulate the initial stages as it develops into an embryo. Later on, the embryo takes control of its own development by destroying these inherited molecules and switches to making its own instead. This process is called the maternal-to-zygotic transition. The molecules passed from the mother to the egg cell include proteins and messenger RNAs (molecules that include the coded instructions to make new proteins). Previous research has begun to reveal how the embryo destroys the mRNAs it inherits from its mother and how it starts to make its own. Yet almost nothing is known about how an embryo gets rid of its mother's proteins. To address this question, Zavortink, Rutt, Dzitoyeva et al. used an approach known as an RNA interference screen to identify factors required to destroy three maternal proteins in fruit fly embryos. The experiments helped identify one enzyme that worked together with another larger enzyme complex to destroy the maternal proteins. This enzyme belongs to a class of enzymes known as ubiquitin-conjugating enzymes (or E2 enzymes) and it was given the name "Kdo", short for "Marie Kondo". Further experiments showed that the mRNAs that code for the Kdo enzyme were present in unfertilized eggs, but in a repressed state that prevented the eggs from making the enzyme. Once an egg started to develop into an embryo, these mRNAs became active and the embryo started to make Kdo enzymes. This led to the three maternal proteins being destroyed during the maternal-to-zygotic transition. These findings reveal a new pathway that regulates the destruction of maternal proteins as the embryo develops. The next challenge will be identifying other maternal proteins that do not "spark joy" and understanding the role their destruction plays in the earliest events of embryonic development.
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Proteínas de Drosophila/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Cigoto/metabolismo , Animales , ARN Helicasas DEAD-box/metabolismo , Drosophila melanogaster/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ribonucleoproteínas/metabolismo , Ubiquitina/metabolismoRESUMEN
A new paradigm has emerged that coding regions can regulate mRNA stability in model organisms. Here, due to differences in cognate tRNA abundance, synonymous codons are translated at different speeds, and slow codons then stimulate mRNA decay. To ask if this phenomenon also occurs in humans, we isolated RNA stability effects due to coding regions using the human ORFeome collection. We find that many open reading frame (ORF) characteristics, such as length and secondary structure, fail to provide explanations for how coding regions alter mRNA stability, and, instead, that the ORF relies on translation to impact mRNA stability. Consistent with what has been seen in other organisms, codon use is related to the effects of ORFs on transcript stability. Importantly, we found instability-associated codons have longer A-site dwell times, suggesting for the first time in humans a connection between elongation speed and mRNA decay. Thus, we propose that codon usage alters decoding speeds and so affects human mRNA stability.
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Codón/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Línea Celular , Células HEK293 , Humanos , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas/genética , Estructura Secundaria de Proteína/genética , ARN de Transferencia/genéticaRESUMEN
In this issue of Cell, Cassidy et al. (2019) show that, in Drosophila melanogaster, developmental abnormalities resulting from loss of repressors such as microRNAs can be suppressed by slow metabolism. They additionally provide insight into the underlying mechanism that connects metabolic state with developmental outcomes.
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Drosophila melanogaster/genética , MicroARNs , Animales , Regulación de la Expresión Génica , Factores de TranscripciónRESUMEN
Communication between the 5' and 3' ends of mature eukaryotic mRNAs lies at the heart of gene regulation, likely arising at the same time as the eukaryotic lineage itself. Our view of how and why it occurs has been shaped by elegant experiments that led to nearly universal acceptance of the "closed-loop model." However, new observations suggest that this classic model needs to be reexamined, revised, and expanded. Here, we address fundamental questions about the closed-loop model and discuss how a growing understanding of mRNA structure, dynamics, and intermolecular interactions presents new experimental opportunities. We anticipate that the application of emerging methods will lead to expanded models that include the role of intrinsic mRNA structure and quantitative dynamic descriptions of 5'-3' proximity linked to the functional status of an mRNA and will better reflect the messy realities of the crowded and rapidly changing cellular environment.