Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5' ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yLuc7 protein (to mimic the lack of Luc7Ls in human U1 snRNP) and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.

Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen.

The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.

Symmetry-breaking malachite green as a near-infrared light-activated fluorogenic photosensitizer for RNA proximity labeling.

Cellular RNA is asymmetrically distributed in cells and the regulation of RNA localization is crucial for proper cellular functions. However, limited chemical tools are available to capture dynamic RNA localization in complex biological systems with high spatiotemporal resolution. Here, we developed a new method for RNA proximity labeling activated by near-infrared (NIR) light, which holds the potential for deep penetration. Our method, termed FAP-seq, utilizes a genetically encoded fluorogen activating protein (FAP) that selectively binds to a set of substrates known as malachite green (MG). FAP binding restricts the rotation of MG and rapidly activates its fluorescence in a wash-free manner. By introducing a monoiodo modification to MG, we created a photosensitizer (MG-HI) with the highest singlet oxygen generation ability among various MG derivatives, enabling both protein and RNA proximity labeling in live cells. New insights are provided in the transcriptome analysis with FAP-seq, while a deeper understanding of the symmetry-breaking structural arrangement of FAP-MG-HI was obtained through molecular dynamics simulations. Overall, our wash-free and NIR light-inducible RNA proximity labeling method (FAP-seq) offers a powerful and versatile approach for investigating complex mechanisms underlying RNA-related biological processes.

Differential subcellular localization of ASPM RNA and protein.

RNAs encoding some centrosomal components are trafficked to the organelle during mitosis. Some RNAs, including ASPM , localize to the centrosome co-translationally. However, the relative position of these RNAs and their protein after trafficking to centrosomes remained unclear. We find that mislocalization of ASPM RNA from the centrosome does not affect the localization of ASPM protein. Further, ASPM RNA and ASPM protein reside in two physically close yet distinct subcellular spaces, with ASPM RNA on the astral side of the centrosome and ASPM protein on the spindle side. This suggests subtly distinct locations of ASPM RNA translation and ASPM protein function.

The Giardia lamblia ribosome structure reveals divergence in several biological pathways and the mode of emetine function.

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.

RNA localization mechanisms transcend cell morphology.

RNA molecules are localized to specific subcellular regions through interactions between RNA regulatory elements and RNA binding proteins (RBPs). Generally, our knowledge of the mechanistic details behind the localization of a given RNA is restricted to a particular cell type. Here, we show that RNA/RBP interactions that regulate RNA localization in one cell type predictably regulate localization in other cell types with vastly different morphologies. To determine transcriptome-wide RNA spatial distributions across the apicobasal axis of human intestinal epithelial cells, we used our recently developed RNA proximity labeling technique, Halo-seq. We found that mRNAs encoding ribosomal proteins (RP mRNAs) were strongly localized to the basal pole of these cells. Using reporter transcripts and single-molecule RNA FISH, we found that pyrimidine-rich motifs in the 5' UTRs of RP mRNAs were sufficient to drive basal RNA localization. Interestingly, the same motifs were also sufficient to drive RNA localization to the neurites of mouse neuronal cells. In both cell types, the regulatory activity of this motif was dependent on it being in the 5' UTR of the transcript, was abolished upon perturbation of the RNA-binding protein LARP1, and was reduced upon inhibition of kinesin-1. To extend these findings, we compared subcellular RNAseq data from neuronal and epithelial cells. We found that the basal compartment of epithelial cells and the projections of neuronal cells were enriched for highly similar sets of RNAs, indicating that broadly similar mechanisms may be transporting RNAs to these morphologically distinct locations. These findings identify the first RNA element known to regulate RNA localization across the apicobasal axis of epithelial cells, establish LARP1 as an RNA localization regulator, and demonstrate that RNA localization mechanisms cut across cell morphologies.

The information required to build a specific protein is encoded into molecules of RNA which are often trafficked to precise locations in a cell. These journeys require a complex molecular machinery to be assembled and set in motion so that the RNA can be transported along dynamic 'roads' called microtubules. The details of this mechanism are known only for a handful of RNAs in a few cell types; for example, scientists have uncovered the signals presiding over the shuttling of certain RNAs to the axon, the long and thin projection that a neuron uses to communicate. Yet these RNAs are also present in cells that lack axons. Whether the molecular processes which preside over RNA movement apply across cell types has so far remained unclear. To investigate this question, Goering et al. tracked the location of RNA molecules in two types of polarized mouse cells: neurons which feature an axon, and 'epithelial' cells which line the intestine. The experiments revealed that the signals sending RNAs to the axons also directed the molecules towards the bottom pole of epithelial cells. In both cases, the RNAs travelled towards the extremity of the growing, "plus" end of the microtubules. Overall, this work suggests that RNA transport mechanisms should not be thought of as leading to a particular location in the cell; instead, they may be following more generalisable instructions. This knowledge could allow scientists to predict where a particular RNA will be sent across cell types based on data from one cell population. It could also aid the development of synthetic RNAs that target specific parts of the cell, offering greater control over their actions.

Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen.

The cytoplasmic dynein-1 (dynein) motor plays a key role in cellular organisation by transporting a wide variety of cellular constituents towards the minus ends of microtubules. However, relatively little is known about how the biosynthesis, assembly and functional diversity of the motor is orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a guide RNA library targeting 18,253 genes, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints generated from multiplexed images revealed co-functional genes involved in many cellular processes, including several candidate novel regulators of core dynein functions. Mechanistic analysis of one of these proteins, the RNA-binding protein SUGP1, provides evidence that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our dataset represents a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organisation that were captured by our high-content imaging.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5' ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in cold-sensitive growth phenotype and moderate splicing defects. Next, we added an auxin-inducible degron to yLuc7 protein and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe 2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondria function in yeast.

Human PRPF39 is an alternative splicing factor recruiting U1 snRNP to weak 5' splice sites.

Human PRPF39 is a homolog of the yeast Prp39 and Prp42 paralogs. We have previously shown that human PRPF39 forms a homodimer that interacts with the CTD of U1C, mirroring the yeast Prp39/Prp42 heterodimer. We demonstrate here that PRPF39 knockdown in HEK293 cells affects many alternative splicing events primarily by reducing the usage of weak 5'ss. Additionally, PRPF39 preferentially binds to a GC-rich RNA, likely at the interface between its NTD and CTD. These data indicate that PRPF39 potentially recruits U1 snRNP to a weak 5' ss, serving as a previously unrecognized alternative splicing factor. We further demonstrate that human TIA1 binds to U1C through its RRM1 and RRM3+Q domains but has no significant binding to PRPF39. Finally, all three human LUC7L isoforms directly interact with U1C. These results reveal significant parallels to the yeast U1 snRNP structure and support the use of yeast U1 snRNP as a model for understanding the mechanism of human alternative splicing.

High-throughput identification of RNA localization elements in neuronal cells.

Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse and human neuronal cells. The localization elements were enriched in adenosine and guanosine residues. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. Depletion of Unk in cells reduced the ability of the elements to drive RNAs to neurites, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons.

Diverse cell-specific patterns of alternative polyadenylation in Drosophila.

Most genes in higher eukaryotes express isoforms with distinct 3' untranslated regions (3' UTRs), generated by alternative polyadenylation (APA). Since 3' UTRs are predominant locations of post-transcriptional regulation, APA can render such programs conditional, and can also alter protein sequences via alternative last exon (ALE) isoforms. We previously used 3'-sequencing from diverse Drosophila samples to define multiple tissue-specific APA landscapes. Here, we exploit comprehensive single nucleus RNA-sequencing data (Fly Cell Atlas) to elucidate cell-type expression of 3' UTRs across >250 adult Drosophila cell types. We reveal the cellular bases of multiple tissue-specific APA/ALE programs, such as 3' UTR lengthening in differentiated neurons and 3' UTR shortening in spermatocytes and spermatids. We trace dynamic 3' UTR patterns across cell lineages, including in the male germline, and discover new APA patterns in the intestinal stem cell lineage. Finally, we correlate expression of RNA binding proteins (RBPs), miRNAs and global levels of cleavage and polyadenylation (CPA) factors in several cell types that exhibit characteristic APA landscapes, yielding candidate regulators of transcriptome complexity. These analyses provide a comprehensive foundation for future investigations of mechanisms and biological impacts of alternative 3' isoforms across the major cell types of this widely-studied model organism.

Halo-seq: An RNA Proximity Labeling Method for the Isolation and Analysis of Subcellular RNA Populations.

The subcellular localization of specific RNA molecules promotes localized cellular activity across a variety of species and cell types. The misregulation of this RNA targeting can result in developmental defects, and mutations in proteins that regulate this process are associated with multiple diseases. For the vast majority of localized RNAs, however, the mechanisms that underlie their subcellular targeting are unknown, partly due to the difficulty associated with profiling and quantifying subcellular RNA populations. To address this challenge, we developed Halo-seq, a proximity labeling technique that can label and profile local RNA content at virtually any subcellular location. Halo-seq relies on a HaloTag fusion protein localized to a subcellular space of interest. Through the use of a radical-producing Halo ligand, RNAs that are near the HaloTag fusion are specifically labeled with spatial and temporal control. Labeled RNA is then specifically biotinylated in vitro via a click reaction, facilitating its purification from a bulk RNA sample using streptavidin beads. The content of the biotinylated RNA is then profiled using high-throughput sequencing. In this article, we describe the experimental and computational procedures for Halo-seq, including important benchmark and quality control steps. By allowing the flexible profiling of a variety of subcellular RNA populations, we envision Halo-seq facilitating the discovery and further study of RNA localization regulatory mechanisms. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualization of HaloTag fusion protein localization Basic Protocol 2: In situ copper-catalyzed cycloaddition of fluorophore via click reaction Basic Protocol 3: In vivo RNA alkynylation and extraction of total RNA Basic Protocol 4: In vitro copper-catalyzed cycloaddition of biotin via click reaction Basic Protocol 5: Assessment of RNA biotinylation by RNA dot blot Basic Protocol 6: Enrichment of biotinylated RNA using streptavidin beads and preparation of RNA-seq library Basic Protocol 7: Computational analysis of Halo-seq data.

Transcriptome-scale methods for uncovering subcellular RNA localization mechanisms.

Across a variety of systems, thousands of RNAs are localized to specific subcellular locations. However, for the vast majority of these RNAs, the mechanisms that underlie their transport are unknown. Historically, these mechanisms were uncovered for a single transcript at a time by laboriously testing the ability of RNA fragments to direct transcript localization. Recently developed methods profile the content of subcellular transcriptomes using high-throughput sequencing, allowing the analysis of the localization of thousands of transcripts at once. By identifying commonalities shared among multiple localized transcripts, these methods have the potential to rapidly expand our understanding of RNA localization mechanisms.

Visualization and Quantification of Subcellular RNA Localization Using Single-Molecule RNA Fluorescence In Situ Hybridization.

Advancements in imaging technologies, especially approaches that allow the imaging of single RNA molecules, have opened new avenues to understand RNA regulation, from synthesis to decay with high spatial and temporal resolution. Here, we describe a protocol for single-molecule fluorescent in situ hybridization (smFISH) using three different approaches for synthesizing the fluorescent probes. The three approaches described are commercially available probes, single-molecule inexpensive FISH (smiFISH), and in-house enzymatically labeled probes. These approaches offer technical and economic flexibility to meet the specific needs of an experiment. In addition, we provide a protocol to perform automated smFISH spot detection using the software FISH-quant.

Analysis of subcellular transcriptomes by RNA proximity labeling with Halo-seq.

Thousands of RNA species display nonuniform distribution within cells. However, quantification of the spatial patterns adopted by individual RNAs remains difficult, in part by a lack of quantitative tools for subcellular transcriptome analysis. In this study, we describe an RNA proximity labeling method that facilitates the quantification of subcellular RNA populations with high spatial specificity. This method, termed Halo-seq, pairs a light-activatable, radical generating small molecule with highly efficient Click chemistry to efficiently label and purify spatially defined RNA samples. We compared Halo-seq with previously reported similar methods and found that Halo-seq displayed a higher efficiency of RNA labeling, indicating that it is well suited to the investigation of small, precisely localized RNA populations. We then used Halo-seq to quantify nuclear, nucleolar and cytoplasmic transcriptomes, characterize their dynamic nature following perturbation, and identify RNA sequence features associated with their composition. Specifically, we found that RNAs containing AU-rich elements are relatively enriched in the nucleus. This enrichment becomes stronger upon treatment with the nuclear export inhibitor leptomycin B, both expanding the role of HuR in RNA export and generating a comprehensive set of transcripts whose export from the nucleus depends on HuR.

LABRAT reveals association of alternative polyadenylation with transcript localization, RNA binding protein expression, transcription speed, and cancer survival.

BACKGROUND: The sequence content of the 3' UTRs of many mRNA transcripts is regulated through alternative polyadenylation (APA). The study of this process using RNAseq data, though, has been historically challenging. RESULTS: To combat this problem, we developed LABRAT, an APA isoform quantification method. LABRAT takes advantage of newly developed transcriptome quantification techniques to accurately determine relative APA site usage and how it varies across conditions. Using LABRAT, we found consistent relationships between gene-distal APA and subcellular RNA localization in multiple cell types. We also observed connections between transcription speed and APA site choice as well as tumor-specific transcriptome-wide shifts in APA isoform abundance in hundreds of patient-derived tumor samples that were associated with patient prognosis. We investigated the effects of APA on transcript expression and found a weak overall relationship, although many individual genes showed strong correlations between relative APA isoform abundance and overall gene expression. We interrogated the roles of 191 RNA-binding proteins in the regulation of APA isoforms, finding that dozens promote broad, directional shifts in relative APA isoform abundance both in vitro and in patient-derived samples. Finally, we find that APA site shifts in the two classes of APA, tandem UTRs and alternative last exons, are strongly correlated across many contexts, suggesting that they are coregulated. CONCLUSIONS: We conclude that LABRAT has the ability to accurately quantify APA isoform ratios from RNAseq data across a variety of sample types. Further, LABRAT is able to derive biologically meaningful insights that connect APA isoform regulation to cellular and molecular phenotypes.

Quantifying alternative polyadenylation in RNAseq data with LABRAT.

Alternative polyadenylation (APA) generates transcript isoforms that differ in their 3' UTR content and may therefore be subject to different regulatory fates. Although the existence of APA has been known for decades, quantification of APA isoforms from high-throughput RNA sequencing data has been difficult. To facilitate the study of APA in large datasets, we developed an APA quantification technique called LABRAT (Lightweight Alignment-Based Reckoning of Alternative Three-prime ends). LABRAT leverages modern transcriptome quantification approaches to determine the relative abundances of APA isoforms. In this manuscript we describe how LABRAT produces its calculations, provide a step-by-step protocol for its use, and demonstrate its ability to quantify APA in single-cell RNAseq data.

Cell-type-specific profiling of human cellular models of fragile X syndrome reveal PI3K-dependent defects in translation and neurogenesis.

Transcriptional silencing of the FMR1 gene in fragile X syndrome (FXS) leads to the loss of the RNA-binding protein FMRP. In addition to regulating mRNA translation and protein synthesis, emerging evidence suggests that FMRP acts to coordinate proliferation and differentiation during early neural development. However, whether loss of FMRP-mediated translational control is related to impaired cell fate specification in the developing human brain remains unknown. Here, we use human patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells and organoids to model neurogenesis in FXS. We developed a high-throughput, in vitro assay that allows for the simultaneous quantification of protein synthesis and proliferation within defined neural subpopulations. We demonstrate that abnormal protein synthesis in FXS is coupled to altered cellular decisions to favor proliferative over neurogenic cell fates during early development. Furthermore, pharmacologic inhibition of elevated phosphoinositide 3-kinase (PI3K) signaling corrects both excess protein synthesis and cell proliferation in a subset of patient neural cells.

ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing.

ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3' UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets of Drosophila are known, ectopic expression of each of the three family members (Elav, Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants of elav/fne, but not elav alone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation of Drosophila ELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3' UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification in Drosophila and mammalian neurons.