Pre-messenger RNA Processing Research Articles

RNA helicase-dependent gene looping impacts messenger RNA processing.

DDX5 and DDX17 are DEAD-box RNA helicase paralogs which regulate several aspects of gene expression, especially transcription and splicing, through incompletely understood mechanisms. A transcriptome analysis of DDX5/DDX17-depleted human cells confirmed the large impact of these RNA helicases on splicing and revealed a widespread deregulation of 3′ end processing. In silico analyses and experiments in cultured cells showed the binding and functional contribution of the genome organizing factor CTCF to chromatin sites at or near a subset of DDX5/DDX17-dependent exons that are characterized by a high GC content and a high density of RNA Polymerase II. We propose the existence of an RNA helicase-dependent relationship between CTCF and the dynamics of transcription across DNA and/or RNA structured regions, that contributes to the processing of internal and terminal exons. Moreover, local DDX5/DDX17-dependent chromatin loops spatially connect RNA helicase-regulated exons with their cognate promoter, and we provide the first direct evidence that de novo gene looping modifies alternative splicing and polyadenylation. Overall our findings uncover the impact of DDX5/DDX17-dependent chromatin folding on pre-messenger RNA processing.

Electron microscopy of nuclear nanoribonucleoproteins (nanoRNPs)

In the cell nucleus several ribonucleoproteins related to gene expression are present The nucleolus coiled bodies and other nuclear particles are among these structures Particles of nanometer size include perichromatin fibrils perichromatin and Balbiani ring granules interchromatin granules and Lacandonia granules All of them are involved in pre messenger RNA intranuclear metabolism Previous and recent research suggests that these particles are involved in transcription and processing of pre messenger RNA within the cell nucleus

Convergent Evidence That ZNF804A Is a Regulator of Pre-messenger RNA Processing and Gene Expression.

Genome-wide association studies have linked common variation in ZNF804A with an increased risk of schizophrenia. However, little is known about the biology of ZNF804A and its role in schizophrenia. Here, we investigate the function of ZNF804A using a variety of complementary molecular techniques. We show that ZNF804A is a nuclear protein that interacts with neuronal RNA splicing factors and RNA-binding proteins including RBFOX1, which is also associated with schizophrenia, CELF3/4, components of the ubiquitin-proteasome system and the ZNF804A paralog, GPATCH8. GPATCH8 also interacts with splicing factors and is localized to nuclear speckles indicative of a role in pre-messenger RNA (mRNA) processing. Sequence analysis showed that GPATCH8 contains ultraconserved, alternatively spliced poison exons that are also regulated by RBFOX proteins. ZNF804A knockdown in SH-SY5Y cells resulted in robust changes in gene expression and pre-mRNA splicing converging on pathways associated with nervous system development, synaptic contact, and cell adhesion. We observed enrichment (P = 1.66 × 10–9) for differentially spliced genes in ZNF804A-depleted cells among genes that contain RBFOX-dependent alternatively spliced exons. Differentially spliced genes in ZNF804A-depleted cells were also enriched for genes harboring de novo loss of function mutations in autism spectrum disorder (P = 6.25 × 10–7, enrichment 2.16) and common variant alleles associated with schizophrenia (P = .014), bipolar disorder and schizophrenia (P = .003), and autism spectrum disorder (P = .005). These data suggest that ZNF804A and its paralogs may interact with neuronal-splicing factors and RNA-binding proteins to regulate the expression of a subset of synaptic and neurodevelopmental genes.

Extremely fast and incredibly close: cotranscriptional splicing in budding yeast.

RNA splicing, an essential part of eukaryotic pre-messenger RNA processing, can be simultaneous with transcription by RNA polymerase II. Here, we compare and review independent next-generation sequencing methods that jointly quantify transcription and splicing in budding yeast. For many yeast transcripts, splicing is fast, taking place within seconds of intron transcription, while polymerase is within a few dozens of nucleotides of the 3′ splice site. Ribosomal protein transcripts are spliced particularly fast and cotranscriptionally. However, some transcripts are spliced inefficiently or mainly post-transcriptionally. Intron-mediated regulation of some genes is likely to be cotranscriptional. We suggest that intermediates of the splicing reaction, missing from current data sets, may hold key information about splicing kinetics.

EPS15R, TASP1, and PRPF3 Are Novel Disease Candidate Genes Targeted by HNF4α Splice Variants in Hepatocellular Carcinomas

The orphan nuclear receptor HNF4alpha is a member of the hepatic transcription factor network. This protein plays a pivotal role in liver development and hepatocellular differentiation. Nine splice variants have been identified, some of which are specifically regulated in disease. The role of HNF4alpha splice variants in hepatocellular carcinomas (HCC) is unknown. Here, we report an identification of novel candidate genes targeted by splice variants of HNF4alpha. We used chromatin immunoprecipitation followed by cloning and sequencing of DNA. Expression of HNF4alpha P1 and P2 promoter-driven isoforms and of genes targeted by HNF4alpha were analyzed by quantitative reverse-transcription polymerase chain reaction, Western blotting, electrophoretic mobility shift assay, and immunohistochemistry. We observed a remarkable switch in gene and protein expression from P1 to P2 promoter-driven fetal isoforms of HNF4alpha in transgenic livers and HCCs of epidermal growth factor (EGF) overexpressing mice and in human HCCs. We further identified EGF-receptor substrate (EPS15R), related EPS15, the premessenger RNA processing factor 3 (PRPF3), and taspase 1 (TASP1) as novel HNF4alpha disease regulated genes with induced expression in mouse and in human HCCs. We suggest EPS15 and EPS15R mediated internalization of activated EGF receptor to result in receptor recycling as to reinforce the proliferative response to EGF. Regulation of the type 2 asparaginase TASP1 and of the splicing factor PRPF3 further documents a switch to fetal liver programs in HCC. We report induction of P2 promoter-driven HNF4alpha splice variants and regulation of disease candidate genes in EGF-induced mouse and human HCC.

PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores

The spindle checkpoint delays anaphase onset until every chromosome kinetochore has been efficiently captured by the mitotic spindle microtubules. In this study, we report that the human pre–messenger RNA processing 4 (PRP4) protein kinase associates with kinetochores during mitosis. PRP4 depletion by RNA interference induces mitotic acceleration. Moreover, we frequently observe lagging chromatids during anaphase leading to aneuploidy. PRP4-depleted cells do not arrest in mitosis after nocodazole treatment, indicating a spindle assembly checkpoint (SAC) failure. Thus, we find that PRP4 is necessary for recruitment or maintenance of the checkpoint proteins MPS1, MAD1, and MAD2 at the kinetochores. Our data clearly identify PRP4 as a previously unrecognized kinetochore component that is necessary to establish a functional SAC.

Pre-messenger RNA processing and its regulation: a genomic perspective.

The sequence of entire genomes from higher eukaryotes revealed that an average gene is very large due to the interruption of the coding sequence with large noncoding introns. Introns are co-transcriptionally removed by splicing with great accuracy and fidelity, although contrary to our expectations, currently known signals required for pre-messenger RNA (mRNA) processing are very degenerate and redundant. Furthermore, the vast majority of genes are alternatively processed. A large number of proteins are therefore involved in generating specificity in pre-mRNA processing that requires a dedicated mechanisms to operate at genomic dimensions. In this review I will summarize recent progress in understanding how established principles of pre-mRNA processing extend to genomic dimensions and discuss emerging concepts in coupling of pre-mRNA processing with other nuclear events and nuclear organization.

RNA editing and alternative splicing: the importance of co‐transcriptional coordination

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (pol II) is essential for several co-transcriptional pre-messenger RNA processing events, including capping, 3'-end processing and splicing. We investigated the role of the CTD of RNA pol II in the coordination of A to I editing and splicing of the ADAR2 (ADAR: adenosine deaminases that act on RNA) pre-mRNA. The auto-editing of Adar2 intron 4 by the ADAR2 adenosine deaminase is tightly coupled to splicing, as the modification of the dinucleotide AA to AI creates a new 3' splice site. Unlike other introns, the CTD is not required for efficient splicing of intron 4 at either the normal 3' splice site or the alternative site created by editing. However, the CTD is required for efficient co-transcriptional auto-editing of ADAR2 intron 4. Our results implicate the CTD in site-selective RNA editing by ADAR2 and in coordination of editing with alternative splicing.

An extensive network of coupling among gene expression machines

Gene expression in eukaryotes requires several multi-component cellular machines. Each machine carries out a separate step in the gene expression pathway, which includes transcription, several pre-messenger RNA processing steps and the export of mature mRNA to the cytoplasm. Recent studies lead to the view that, in contrast to a simple linear assembly line, a complex and extensively coupled network has evolved to coordinate the activities of the gene expression machines. The extensive coupling is consistent with a model in which the machines are tethered to each other to form 'gene expression factories' that maximize the efficiency and specificity of each step in gene expression.

Pre-messenger RNA processing factors in the Drosophila genome.

In eukaryotes, messenger RNAs are generated by a process that includes coordinated splicing and 3′ end formation. Factors essential for the splicing of mRNA precursors (pre-mRNA) in eukaryotes have been identified primarily through the study of nuclear extracts derived from mammalian cells and

The 1.7 A crystal structure of the regulator of chromosome condensation (RCC1) reveals a seven-bladed propeller.

The gene encoding the regulator of chromosome condensation (RCC1) was cloned by virtue of its ability to complement the temperature-sensitive phenotype of the hamster cell line tsBN2, which undergoes premature chromosome condensation or arrest in the G1 phase of the cell cycle at non-permissive temperatures. RCC1 homologues have been identified in many eukaryotes, including budding and fission yeast. Mutations in the gene affect pre-messenger RNA processing and transport, mating, initiation of mitosis and chromatin decondensation, suggesting that RCC1 is important in the control of nucleo-cytoplasmic transport and the cell cycle. Biochemically, RCC1 is a guanine-nucleotide-exchange factor for the nuclear Ras homologue Ran; it increases the dissociation of Ran-bound GDP by 10(5)-fold. It may also bind to DNAvia a protein-protein complex. Here we show that the structure of human RCC1, solved to 1.7-A resolution by X-ray crystallography, consists of a seven-bladed propeller formed from internal repeats of 51-68 residues per blade. The sequence and structure of the repeats differ from those of WD40-domain proteins, which also form seven-bladed propellers and include the beta-subunits of G proteins. The nature of the structure explains the consequences of a wide range of known mutations. The region of the protein that is involved in guanine-nucleotide exchange is located opposite the region that is thought to be involved in chromosome binding.

An alternative molecular mechanism of action of 5-fluorouracil, a potent anticancer drug

It is assumed that the primary mode of action of 5-fluorouracil (5-FUra) is mediated via inhibition of thymidylate synthetase. Persistent inhibition of cellular proliferation after treatment of the 5-FUra-inhihited cells with exogenous thymidine do not support the notion that the anti-proliferative action of 5-FUra is due exclusively to inhibition of DNA replication. Our studies have revealed an alternative mechanism of action at the level of pre-ribosomal RNA (pre-rRNA) processing. Pre-rRNA processing was inhibited completely in vivo as well as in S-100 extract from the mouse lymphosarcoma P1798 cells that were treated with 5-FUra. Under this condition, the 5-FUra-substituted pre-rRNA substrate was processed efficiently at the primary processing site. This study showed that the activity and/or the synthesis of a factor potentially involved in pre-rRNA processing is blocked in cells treated with the fluoropyrimidine. UV-cross-linking study showed that a 200 kDa polypeptide designated ribosomal RNA binding protein (RRBP) was absent in the S-100 extract from the drug-treated mouse lymphosarcoma cells. Since a polypeptide that cross-links to a processing site on RNA is usually involved in the RNA processing, RRBP may have a direct role in pre-rRNA processing. A key molecular mechanism for the antiproliferative action of 5-FUra may be due to its interference with the activity and/or synthesis of RRBP. Exposure of cells to 5-FUra did not inhibit the interaction between U3 small nucleolar RNA (snoRNA) and pre-rRNA at the primary processing site (a key step in the processing reaction) and the formation of U3 small nucleolar ribonucleoprotein (snoRNP). Treatment of cells with the fluoropyrimidine did not block the 3′ end processing of pre-messenger RNA (pre-mRNA). This article also discusses the effects of 5-FUra on pre-mRNA splicing and mRNA translation, and proposes other avenues of research to explore further the mechanism of action of this important pyrimidine analog. biochem pharmacol 53;11:1569-1575, 1997.

Intranuclear foci containing low abundance herpes simplex virus latency-associated transcripts visualized by non-isotopic in situ hybridization.

During latent infection of neurons with herpes simplex virus type 1 (HSV-1), several RNA transcripts of varying abundance arise from a single locus within the virus repeats. The functions of latency-associated transcripts (LATs) are unknown and the relationship between the various RNA species requires further clarification. Reported here is a novel approach to the study of HSV transcripts during latency, based on the increasing realization that cellular and viral RNAs are synthesized and processed by macromolecular complexes that occupy discrete compartments within the nucleoplasm of a cell. High resolution non-isotopic in situ hybridization was used to study the intranuclear topology of HSV-1 LATs in primary sensory neurons of latently infected mice and humans. Low abundance (minor) LATs were localized to sharply defined intranuclear foci of 1 to 3 microns in diameter. On average, there were 2.6 to 2.8 foci/LAT+ neuronal profile (5 microns), representing 13 to 14 foci/cell. In contrast to the focal deployment of minor LATs, the more abundant latency-associated RNAs were distributed diffusely throughout the nucleoplasms of latency infected neurons, with prominent sparing of nucleolar regions. These data establish a foundation for studying the synthesis, processing and transport of LATs in vivo. It should now be possible to investigate the nature of those cellular products which associate with HSV-1 encoded LATs in vivo and thereby determine whether minor LATs are associated with previously characterized macromolecular complexes, such as those responsible for processing of pre-messenger RNA.

Ultrastructural analysis of early pre-messenger RNA processing events using the Miller chromatin spreading method

We use the Miller chromatin spreading technique for electron microscopic visualization of transcriptionally active genes. In this method, cells are hypotonically disrupted and cellular contents are diluted into water at pH 8-9 and fixed with formaldehyde. The dispersed cellular contents are centrifuged onto a carbon-coated EM grid; the majority of the material that is deposited on the grid consists of entangled masses of dispersed chromatin, some regions of which are transcriptionally active (Fig 1). Our interests lie in ultrastructural analysis of co-transcriptional RNA processing events on pre-mRNA transcripts, which we analyze by mapping structural features on successive nascent transcripts on a given gene. The two processing events that we have been able to study by this approach are the removal of introns by splicing and generation of the 3’ end of the transcript.

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins.

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.

Primary and secondary structure of dinoflagellate U5 small nuclear RNA.

U5 RNA is one of the six capped small nuclear RNAs present in most eukaryotic cells. Like U1, U2, U4 and U6 RNAs, U5 RNA is associated with hnRNP particles and is thus probably involved in some, as yet undefined, aspects of pre-messenger RNA processing. In this study, the complete nucleotide sequence of U5 RNA of a dinoflagellate, Crypthecodinium cohnii was determined. The analysis of this dinoflagellate U5 RNA sequence showed that a) the sequence homology between human, rat and chicken U5 RNA sequences and dinoflagellate U5 RNA sequence is 64%; b) the extent and the position of post-transcriptional modifications are similar to those found in U5 RNA of higher eukaryotes; c) although the dinoflagellate U5 RNA is shorter in length (108 nucleotides long vs 117 long in human, rat and chicken cells), the RNA fits well into the same secondary structure proposed for U5 RNA of higher eukaryotes (Krol et al. (1981) Nucl. Acids Res. 9, 769); and d) the AUn nucleotide sequence protected by the Sm-antigen and the tight secondary structure found near the 3'-end of other U-RNAs was also found in dinoflagellate U5 RNA. The high order of homology observed between dinoflagellate U5 RNA and U5 RNA of higher eukaryotes indicates that dinoflagellates are more closely related to metazoans than to early eukaryotes.

Physiological adaptation at the molecular level: the frontier where research on differentiation and malignancy meet.

When Walter Cannon wrote Wisdom ofthe Body in 1932 (1), almost nothing was known about the detailed pathways of intermediary metabolism or about intracellular enzymes and their control. Cannon's holistic pointofview emphasizing homeostasis was a brilliant synthesisof the contributions of Claude Bernard, as well as Richet, Pfluger, Fre- dericq, and others, but it has been waiting too long for a new synthesis at the molecular level. This view reiterates a point well stated by Eugene Knox, who coined the phrase enzyme physiology. Recently, Knox ed- itorialized on Antizymes, Adaptation and Homeostasis (2) and com- mented diat the concept ofhomeostasis has often functioned as a heuristic device for relating regulatory factors that were already known. It focussed on stability and constancy of the organism. .. . new regulatory science has not ignored constancy, but its emphasis has been on the change that must accompany constancy. larger theme is con- tinuity of life through purposive change. Such a thought is contained in the world, adaptation, a concept much used by Darwin and the evolu- tionists and an older concept than homeostasis. Knox continued, There is needagainforagrand conceptualscheme that will integrate what some knowas homeostasis with the related but newer molecufar discoveries ofgene expression, This lecture was given at the meeting of the Southwest Section ofthe American Associ- ation for Cancer Research, Albuquerque, New Mexico, November 15-16, 1979, at which time the author was presented the Fourth Annual Noble Foundation Research Recogni- tion Award. Work in the author's laboratory has been supported in part by grants CA- 07175, CA-22484, and CA-17334 from the National Cancer Institute. Since this manu- script was prepared, new advances have indicated that the processing of premessenger RNA is still another component ofphysiological regulation at the molecular level. Some of the new developments have been covered in a more recent lecture, The Present Statusof the Blocked Ontogeny Hypothesis ofNeoplasia: Thalassemia Connection (to appear

Small RNAs in HnRNP fibrils and their possible function in splicing.

Several arguments are in favor of a function of snRNA in the processing of premessenger RNA. A large fraction of snRNA is localized in hnRNP which are assumed to be the site of processing. The different snRNA species are not bound to hnRNP in a unique manner but are associated with both proteins and hnRNA which suggests the possibility of metabolic exchanges in the course of processing. There is approximately 1-2 molecules of snRNA per individual hnRNP. We reexamined the possibility that U1A RNA might serve for the alignment of the extremities of the intron sequences of premessenger RNA insuring correct condition for cutting and splicing. We found that only a UCCA (3' leads to 5') sequence at position 8-11 of U1A RNA was complementary to an AG-GU (5' leads to 3') around a putative splice point for 69 different introns sequenced so far. On the basis of secondary structure of U1A RNA, the UCCA sequence would be available for hybridization. The UCCA sequence is also present in U2 RNA and 4.5 S RNAI. It might associate with AG-GU in a manner similar to that of codon-anticodon, the stability of the complex being insured by the configuration of hnRNP. The possible formation of larger hybrids stable by themselves is unlikely upon examination of the nucleotide sequence of various introns adjacent to the splice point. As there is no direct experimental evidence for the function of snRNA in splicing, there considerations are speculative at the present time. The possibility that adenovirus encoded VA RNA would play a role in splicing was also examined. Various arguments suggest that this possibility is rather remote.

The Nuclear Particles from Rat Brain and Liver are Similar but not Identical

Summary The structure and protein composition of nuclear particles from rat liver were compared to that of rat brain previously described. Many similarities were found. (1) The liver particles were made of units (30–50 S monoparticles α and β, complexes heterogeneous in size, up to 200 S) with the same characteristics as described previously for brain [12]. (2) The overall protein compositions of the particles were similar and most of the major proteins were common to the two tissues. (3) The amount of a few proteins varied with the sedimentation coefficient of the particles in both tissues. The results suggest that the major particle proteins play a general role in the processing of premessenger RNA. However, the particles were not identical. The major difference lay in the quantitative distribution of the common proteins, suggesting that the composition of the constitutive units could vary, perhaps in relation with regulatory processes.