Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data

RanBP2/Nup358 Potentiates the Translation of a Subset of mRNAs Encoding Secretory Proteins

In higher eukaryotes, most mRNAs that encode secreted or membrane-bound proteins contain elements that promote an alternative mRNA nuclear export (ALREX) pathway. Here we report that ALREX-promoting elements also potentiate translation in the presence of upstream nuclear factors. These RNA elements interact directly with, and likely co-evolved with, the zinc finger repeats of RanBP2/Nup358, which is present on the cytoplasmic face of the nuclear pore. Finally we show that RanBP2/Nup358 is not only required for the stimulation of translation by ALREX-promoting elements, but is also required for the efficient global synthesis of proteins targeted to the endoplasmic reticulum (ER) and likely the mitochondria. Thus upon the completion of export, mRNAs containing ALREX-elements likely interact with RanBP2/Nup358, and this step is required for the efficient translation of these mRNAs in the cytoplasm. ALREX-elements thus act as nucleotide platforms to coordinate various steps of post-transcriptional regulation for the majority of mRNAs that encode secreted proteins

Matrin3: connecting gene expression with the nuclear matrix.

As indicated by its name, Matrin3 was discovered as a component of the nuclear matrix, an insoluble fibrogranular network that structurally organizes the nucleus. Matrin3 possesses both DNA- and RNA-binding domains and, consistent with this, has been shown to function at a number of stages in the life cycle of messenger RNAs. These numerous activities indicate that Matrin3, and indeed the nuclear matrix, do not just provide a structural framework for nuclear activities but also play direct functional roles in these activities. Here, we review the structure, functions, and molecular interactions of Matrin3 and of Matrin3-related proteins, and the pathologies that can arise upon mutation of Matrin3. WIREs RNA 2016, 7:303-315. doi: 10.1002/wrna.1336 For further resources related to this article, please visit the WIREs website.We thank Clare Gooding and Dipen Rajgor for critical comments on the manuscript. Work in the CWJS lab on Matrin3 is funded by a grant from the Wellcome Trust (092900). JA was funded by a Boehringer Ingelheim Fonds studentship.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/wrna.133

Genome Analysis Reveals Interplay between 5′UTR Introns and Nuclear mRNA Export for Secretory and Mitochondrial Genes

In higher eukaryotes, messenger RNAs (mRNAs) are exported from the nucleus to the cytoplasm via factors deposited near the 5′ end of the transcript during splicing. The signal sequence coding region (SSCR) can support an alternative mRNA export (ALREX) pathway that does not require splicing. However, most SSCR–containing genes also have introns, so the interplay between these export mechanisms remains unclear. Here we support a model in which the furthest upstream element in a given transcript, be it an intron or an ALREX–promoting SSCR, dictates the mRNA export pathway used. We also experimentally demonstrate that nuclear-encoded mitochondrial genes can use the ALREX pathway. Thus, ALREX can also be supported by nucleotide signals within mitochondrial-targeting sequence coding regions (MSCRs). Finally, we identified and experimentally verified novel motifs associated with the ALREX pathway that are shared by both SSCRs and MSCRs. Our results show strong correlation between 5′ untranslated region (5′UTR) intron presence/absence and sequence features at the beginning of the coding region. They also suggest that genes encoding secretory and mitochondrial proteins share a common regulatory mechanism at the level of mRNA export

RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease

To facilitate precision medicine and whole genome annotation, we developed a machine learning technique that scores how strongly genetic variants affect RNA splicing, whose alteration contributes to many diseases. Analysis of over 650,000 intronic and exonic variants reveals widespread patterns of mutation-driven aberrant splicing. Intronic disease mutations alter splicing nine times more often than common variants, and missense exonic disease mutations that least impact protein function are five times more likely to alter splicing than others. Tens of thousands of disease-causing mutations are detected, including those involved in cancers and spinal muscular atrophy. Examination of intronic and exonic variants found using whole genome sequencing of individuals with autism reveals mis-spliced genes with neurodevelopmental phenotypes. Our approach provides evidence for causal variants and should enable new discoveries in precision medicine

Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON Delta 165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.Institute of Molecular Biology Core Facilities; DFG [ZA 881/2-1, KO 4566/4-1, LE 3473/2-1]; LOEWE program Ubiquitin Networks (Ub-Net) of the State of Hesse (Germany); Deutsche Forschungsgemeinschaft [SFB902 B13]; EMBO [3057]; Fundacao para a Ciencia e a Tecnologia, Portugal (FCT Investigator Starting Grant) [IF/00595/2014]; German Federal Ministry of Research (BMBF; e:bio junior group program) [FKZ: 0316196]; Boehringer Ingelheim Foundation; [INST 47/870-1 FUGG

Unexpected similarities between C9ORF72 and sporadic forms of ALS/FTD suggest a common disease mechanism.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of a disease spectrum with shared clinical, genetic and pathological features. These include near ubiquitous pathological inclusions of the RNA-binding protein (RBP) TDP-43, and often the presence of a GGGGCC expansion in the C9ORF72 (C9) gene. Previously, we reported that the sequestration of hnRNP H altered the splicing of target transcripts in C9ALS patients (Conlon et al., 2016). Here, we show that this signature also occurs in half of 50 postmortem sporadic, non-C9 ALS/FTD brains. Furthermore, and equally surprisingly, these 'like-C9' brains also contained correspondingly high amounts of insoluble TDP-43, as well as several other disease-related RBPs, and this correlates with widespread global splicing defects. Finally, we show that the like-C9 sporadic patients, like actual C9ALS patients, were much more likely to have developed FTD. We propose that these unexpected links between C9 and sporadic ALS/FTD define a common mechanism in this disease spectrum.This work was supported by NIH grant R35 GM 118136 to JLM. EGC was supported in part by NIH training grant 5T32GM008798. RNA sequencing and related analyses at the NYGC were supported by the ALS Association (grant 15-LGCA-234) and the Tow Foundation, which also provided direct support for the Eleanor and Lou Gehrig ALS Center (NAS)

Functional Consequences of Mammalian-Specific Alternative Splicing Events in RNA Binding Proteins

Alternative splicing (AS) greatly increases transcriptome diversity by allowing individual transcripts to be processed into multiple mature RNAs. Advances in RNA sequencing technology have revealed that transcripts from nearly all multi-exon genes in vertebrates are subject to AS, and that patterns of AS change during development and in response to environmental stimuli and disease. Transcriptomic analyses of diverse vertebrate species have provided global insight into AS evolution, revealing an expanded role for this process in mammals and showing that AS patterns have diverged more rapidly than changes in gene expression. While these studies uncovered an extensive number of species- and lineage-specific splice variants, the molecular function of these isoforms and their contribution to phenotypic change remain largely unexplored. In this thesis, I describe a detailed investigation of the functional consequences of splice isoform evolution in multiple RNA binding proteins. I show that mammalian-specific skipping of exon 9 in the PTBP1 splicing factor alters its regulatory activity, and engineered skipping of the orthologous exon in chicken is sufficient to induce a large number of mammalian-like changes in the transcriptome. The inclusion of this exon is regulated during neurogenesis, and significantly impacts the kinetics of activation of neural-specific splicing programs. Subsequent analysis of all mammalian-specific AS events revealed that they are enriched among glycine- and tyrosine-rich, intrinsically disordered protein domains. Such exons affect nearly all members of the heterogeneous nuclear ribonucleoprotein (hnRNP) A and D protein family members, which have diverse functions in RNA biology. At the transcript level, their regulation requires formation of long-range, intramolecular, mammalian-specific RNA duplexes. At the protein level, their inclusion facilitates higher-order hnRNP assemblies on substrate pre-mRNAs that are required for regulation of target AS events. Together, my thesis work demonstrates how splice isoform evolution in RNA binding proteins can expand the regulatory capabilities of mammalian cells, and reveals how these changes can influence evolution of developmental processes.Ph.D