An unexpected path for Malat1 in neurons: trafficking out of the nucleus for translation.

The Malat1 (metastasis-associated lung adenocarcinoma transcript 1) long noncoding RNA is highly and broadly expressed in mammalian tissues, accumulating in the nucleus where it modulates expression and pre-mRNA processing of many protein-coding genes. In this issue of Genes & Development, Xiao and colleagues (doi:10.1101/gad.351557.124) report that a significant fraction of Malat1 transcripts in cultured mouse neurons are surprisingly exported from the nucleus. These transcripts are packaged with Staufen proteins in RNA granules and traffic down the lengths of neurites. They then can be released in a stimulus-dependent manner to be locally translated into a microprotein that alters neuronal gene expression patterns.

Overlapping genes in natural and engineered genomes.

Modern genome-scale methods that identify new genes, such as proteogenomics and ribosome profiling, have revealed, to the surprise of many, that overlap in genes, open reading frames and even coding sequences is widespread and functionally integrated into prokaryotic, eukaryotic and viral genomes. In parallel, the constraints that overlapping regions place on genome sequences and their evolution can be harnessed in bioengineering to build more robust synthetic strains and constructs. With a focus on overlapping protein-coding and RNA-coding genes, this Review examines their discovery, topology and biogenesis in the context of their genome biology. We highlight exciting new uses for sequence overlap to control translation, compress synthetic genetic constructs, and protect against mutation.

IRE1α Mediates the Hypertrophic Growth of Cardiomyocytes Through Facilitating the Formation of Initiation Complex to Promote the Translation of TOP-Motif Transcripts.

BACKGROUND: Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the unfolded protein response transducers and effectors directly take part in the control of protein synthesis. The connection between critical functions of the unfolded protein response in cellular physiology and requirements of multiple processes for cell growth prompted us to investigate the role of the unfolded protein response in cell growth and underlying molecular mechanisms. METHODS: Cardiomyocyte-specific inositol-requiring enzyme 1α (IRE1α) knockout and overexpression mouse models were generated to explore its function in vivo. Neonatal rat ventricular myocytes were isolated and cultured to evaluate the role of IRE1α in cardiomyocyte growth in vitro. Mass spectrometry was conducted to identify novel interacting proteins of IRE1α. Ribosome sequencing and polysome profiling were performed to determine the molecular basis for the function of IRE1α in translational control. RESULTS: We show that IRE1α is required for cell growth in neonatal rat ventricular myocytes under prohypertrophy treatment and in HEK293 cells in response to serum stimulation. At the molecular level, IRE1α directly interacts with eIF4G and eIF3, 2 critical components of the translation initiation complex. We demonstrate that IRE1α facilitates the formation of the translation initiation complex around the endoplasmic reticulum and preferentially initiates the translation of transcripts with 5' terminal oligopyrimidine motifs. We then reveal that IRE1α plays an important role in determining the selectivity and translation of these transcripts. We next show that IRE1α stimulates the translation of epidermal growth factor receptor through an unannotated terminal oligopyrimidine motif in its 5' untranslated region. We further demonstrate a physiological role of IRE1α-governed protein translation by showing that IRE1α is essential for cardiomyocyte growth and cardiac functional maintenance under hemodynamic stress in vivo. CONCLUSIONS: These studies suggest a noncanonical, essential role of IRE1α in orchestrating protein synthesis, which may have important implications in cardiac hypertrophy in response to pressure overload and general cell growth under other physiological and pathological conditions.

Proteome profiling of Pseudomonas aeruginosa PAO1 identifies novel responders to copper stress.

BACKGROUND: The opportunistic pathogen, Pseudomonas aeruginosa is well known for its environmental and metabolic versatility, yet many of the functions of its gene-products remain to be fully elucidated. This study's objective was to illuminate the potential functions of under-described gene-products during the medically relevant copper-stress condition. RESULTS: We used data-independent acquisition mass spectrometry to quantitate protein expression changes associated with copper stress in P. aeruginosa PAO1. Approximately 2000 non-redundant proteins were quantified, with 78 proteins altering in abundance by +/- 1.5-fold or more when cultured to mid-log growth in the presence of 50 µM copper sulfate. One-third of those differentially expressed proteins have no prior established functional roles. CONCLUSIONS: This study provides evidence for the functional involvement of some specific proteins in enabling P. aeruginosa to survive under sub-lethal concentrations of copper. This further paves the way for targeted investigations into the specific mechanisms of their activity.

IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication.

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ϕX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ϕX174 replication; moreover, their absence has no discernible effect on ϕX174 fecundity. These results suggest IbpA/B upregulation is a response to ϕX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.

The dark proteome: translation from noncanonical open reading frames.

Omics-based technologies have revolutionized our understanding of the coding potential of the genome. In particular, these studies revealed widespread unannotated open reading frames (ORFs) throughout genomes and that these regions have the potential to encode novel functional (micro-)proteins and/or hold regulatory roles. However, despite their genomic prevalence, relatively few of these noncanonical ORFs have been functionally characterized, likely in part due to their under-recognition by the broader scientific community. The few that have been investigated in detail have demonstrated their essentiality in critical and divergent biological processes. As such, here we aim to discuss recent advances in understanding the diversity of noncanonical ORFs and their roles, as well as detail biologically important examples within the context of the mammalian genome.

Proteomic and Transcriptomic Analysis of Microviridae φX174 Infection Reveals Broad Upregulation of Host Escherichia coli Membrane Damage and Heat Shock Responses.

Measuring host-bacteriophage dynamics is an important approach to understanding bacterial survival functions and responses to infection. The model Microviridae bacteriophage φX174 is endemic to the human gut and has been studied for over 70 years, but the host response to infection has never been investigated in detail. To address this gap in our understanding of this important interaction within our microbiome, we have measured host Escherichia coli C proteomic and transcriptomic response to φX174 infection. We used mass spectrometry and RNA sequencing (RNA-seq) to identify and quantify all 11 φX174 proteins and over 1,700 E. coli proteins, enabling us to comprehensively map host pathways involved in φX174 infection. Most notably, we see significant host responses centered on membrane damage and remodeling, cellular chaperone and translocon activity, and lipoprotein processing, which we speculate is due to the peptidoglycan-disruptive effects of the φX174 lysis protein E on MraY activity. We also observe the massive upregulation of small heat shock proteins IbpA/B, along with other heat shock pathway chaperones, and speculate on how the specific characteristics of holdase protein activity may be beneficial for viral infections. Together, this study enables us to begin to understand the proteomic and transcriptomic host responses of E. coli to Microviridae infections and contributes insights to the activities of this important model host-phage interaction.IMPORTANCE A major part of the healthy human gut microbiome is the Microviridae bacteriophage, exemplified by the model φX174 phage, and their E. coli hosts. Although much has been learned from studying φX174 over the last half-century, until this work, the E. coli host response to infection has never been investigated in detail. We reveal the proteomic and transcriptomic pathways differentially regulated during the φX174 infection cycle and uncover the details of a coordinated cellular response to membrane damage that results in increased lipoprotein processing and membrane trafficking, likely due to the phage antibiotic-like lysis protein. We also reveal that small heat shock proteins IbpA/B are massively upregulated during infection and that these holdase chaperones are highly conserved across the domains of life, indicating that reliance on them is likely widespread across viruses.

Genome Modularization Reveals Overlapped Gene Topology Is Necessary for Efficient Viral Reproduction.

Sequence overlap between two genes is common across all genomes, with viruses having high proportions of these gene overlaps. Genome modularization and refactoring is the process of disrupting natural gene overlaps to separate coding sequences to enable their individual manipulation. The biological function and fitness effects of gene overlaps are not fully understood, and their effects on gene cluster and genome-level refactoring are unknown. The bacteriophage φX174 genome has ∼26% of nucleotides involved in encoding more than one gene. In this study we use an engineered φX174 phage containing a genome with all gene overlaps removed to show that gene overlap is critical to maintaining optimal viral fecundity. Through detailed phenotypic measurements we reveal that genome modularization in φX174 causes virion replication, stability, and attachment deficiencies. Quantitation of the complete phage proteome across an infection cycle reveals 30% of proteins display abnormal expression patterns. Taken together, we have for the first time comprehensively demonstrated that gene modularization severely perturbs the coordinated functioning of a bacteriophage replication cycle. This work highlights the biological importance of gene overlap in natural genomes and that reducing gene overlap disruption should be an integral part of future genome engineering projects.

Measuring Amber Initiator tRNA Orthogonality in a Genomically Recoded Organism.

Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E. coli strain C321.ΔA. exp lacking all UAG stop codons was created, freeing this "amber" stop codon for other purposes. An engineered "amber initiator" tRNACUAfMet that activates translation at UAG codons is available, but little is known about this tRNA's orthogonality. Here, we combine for the first time the amber initiator tRNACUAfMet in C321.ΔA. exp and measure its cellular effects. We found that the tRNACUAfMet expression resulted in a nearly 200-fold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosome-associated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for off-target translation initiation. In contrast to previous work, we show that UAG-initiated proteins carry N-terminal methionine, but have no evidence for glutamine. Together, our results identify beneficial features of using the amber initiator tRNACUAfMet to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.