The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living ...cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.
Display omitted
•An optimized CRISPR enables live imaging and better gene regulation in human cells•CRISPR imaging visualizes either repetitive or nonrepetitive genomic sequences•CRISPR imaging reports telomere length change and telomere movements•CRISPR imaging monitors the dynamics of gene loci throughout the cell cycle
A new CRISPR-based technology allows precise visualization of individual gene loci in living cells.
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based ...on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
Display omitted
► Inactive CRISPR associated 9 protein (dCas9) is repurposed for genome engineering ► dCas9 and a complementary short guide RNA can target specific genomic sites ► CRISPR interference (CRISPRi) can regulate multiple genes without off-target effects ► CRISPRi is compact and can be ported to bacterial and mammalian cells
The authors have developed a CRISPR interference system in which a catalytically dead Cas9 protein can be targeted to a specific genomic site through a complementary small guide RNA, allowing systematic perturbation of gene transcription in bacteria and mammalian cells.
The signal recognition particle (SRP) enables cotranslational delivery of proteins for translocation into the endoplasmic reticulum (ER), but its full in vivo role remains incompletely explored. We ...combined rapid auxin-induced SRP degradation with proximity-specific ribosome profiling to define SRP's in vivo function in yeast. Despite the classic view that SRP recognizes amino-terminal signal sequences, we show that SRP was generally essential for targeting transmembrane domains regardless of their position relative to the amino terminus. By contrast, many proteins containing cleavable amino-terminal signal peptides were efficiently cotranslationally targeted in SRP's absence. We also reveal an unanticipated consequence of SRP loss: Transcripts normally targeted to the ER were mistargeted to mitochondria, leading to mitochondrial defects. These results elucidate SRP's essential roles in maintaining the efficiency and specificity of protein targeting.
As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global ...monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the
Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, β-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ∼100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors.
Display omitted
Display omitted
► Ribosome profiling broadly enables quantitative analysis of translation in bacteria ► Selective ribosome profiling reveals cotranslational chaperone action of trigger factor ► Trigger factor engages nascent chains only after ∼100 residues have been translated ► Outer-membrane porins are highly enriched among trigger factor substrates
Ribosome profiling offers a dynamic view of a chaperone's engagement with nascent polypeptides, yielding insights into its selective activity that have been opaque to more traditional biochemical and genetic analyses.
Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively ...measure translation at the mitochondrial surface in yeast. Most inner-membrane proteins were cotranslationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of proteins were targeted to a specific organelle. A prominent exception was the fumarate reductase Osm1, known to reside in mitochondria. We identified a conserved ER isoform of Osm1, which contributes to the oxidative protein-folding capacity of the organelle. This dual localization was enabled by alternative translation initiation sites encoding distinct targeting signals. These findings highlight the exquisite in vivo specificity of organellar targeting mechanisms.
Stochasticity is a hallmark of cellular processes, and different classes of genes show large differences in their cell-to-cell variability (noise). To decipher the sources and consequences of this ...noise, we systematically measured pairwise correlations between large numbers of genes, including those with high variability. We find that there is substantial pathway variability shared across similarly regulated genes. This induces quantitative correlations in the expression of functionally related genes such as those involved in the Msn2/4 stress response pathway, amino-acid biosynthesis, and mitochondrial maintenance. Bioinformatic analyses and genetic perturbations suggest that fluctuations in PKA and Tor signaling contribute to pathway-specific variability. Our results argue that a limited number of well-delineated “noise regulons” operate across a yeast cell and that such coordinated fluctuations enable a stochastic but coherent induction of functionally related genes. Finally, we show that pathway noise is a quantitative tool for exploring pathway features and regulatory relationships in un-stimulated systems.
► Pathway noise impacts S. cerevisiae stress and amino acid biosynthesis genes ► Single cell covariance measurements reveal functionally coherent noise regulons ► Genetic perturbations point to PKA and TOR as significant sources of fluctuations ► Correlated noise is a quantitative tool to probe regulatory relationships
Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in ...microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.
Display omitted
► Ultracomplex shRNA library minimizes false positives/negatives in genome-wide screens ► Pooled double-shRNA strategy systematically maps genetic interactions between hits ► Application of two-step strategy identifies pathways controlling ricin susceptibility ► The resulting map uncovers functionally distinct mammalian TRAPP complexes
A high-throughput method that relies on the use of ultracomplex shRNA libraries makes it possible to create genetic interaction maps in mammalian cells. This approach will be applicable to many cellular processes and conditions, as illustrated by the discovery of distinct TRAPP complexes involved in endocytosis.
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.
Mounting evidence of widespread noncanonical translation suggests that the protein-coding potential of the human genome has been underestimated.Recent advances in techniques to probe the translatome and proteome have highlighted noncanonical open reading frames (ORFs) that (i) govern translational regulation, (ii) produce functional microproteins, and/or (iii) encode protein variants of the canonical coding sequence.There are accumulating examples within the literature of noncanonical ORF-encoded proteins that possess important functional and physiological roles, but there lie thousands of putative ORFs within ‘omics’ databases that are left unverified and functionally uncharacterized.
The unfolded protein response (UPR) allows the endoplasmic reticulum (ER) to recover from the accumulation of misfolded proteins, in part by increasing its folding capacity. Inositol-requiring ...enzyme-1 (IRE1) promotes this remodeling by detecting misfolded ER proteins and activating a transcription factor, X-box-binding protein 1, through endonucleolytic cleavage of its messenger RNA (mRNA). Here, we report that IRE1 independently mediates the rapid degradation of a specific subset of mRNAs, based both on their localization to the ER membrane and on the amino acid sequence they encode. This response is well suited to complement other UPR mechanisms because it could selectively halt production of proteins that challenge the ER and clear the translocation and folding machinery for the subsequent remodeling process.
Some nascent proteins that fold within the endoplasmic reticulum (ER) never reach their native state. Misfolded proteins are removed from the folding machinery, dislocated from the ER into the ...cytosol, and degraded in a series of pathways collectively referred to as ER-associated degradation (ERAD). Distinct ERAD pathways centered on different E3 ubiquitin ligases survey the range of potential substrates. We now know many of the components of the ERAD machinery and pathways used to detect substrates and target them for degradation. Much less is known about the features used to identify terminally misfolded conformations and the broader role of these pathways in regulating protein half-lives.
PDF
