U12-tyypin spliseosomi : lokalisaatio ja lähetti-RNA:n silmukoinnin tehokkuuden vaikutukset geenitoimintaan

The removal of non-coding sequences, introns, is an essential part of messenger RNA processing. In most metazoan organisms, the U12-type spliceosome processes a subset of introns containing highly conserved recognition sequences. U12-type introns constitute less than 0,5% of all introns and reside preferentially in genes related to information processing functions, as opposed to genes encoding for metabolic enzymes. It has previously been shown that the excision of U12-type introns is inefficient compared to that of U2-type introns, supporting the model that these introns could provide a rate-limiting control for gene expression. The low efficiency of U12-type splicing is believed to have important consequences to gene expression by limiting the production of mature mRNAs from genes containing U12-type introns. The inefficiency of U12-type splicing has been attributed to the low abundance of the components of the U12-type spliceosome in cells, but this hypothesis has not been proven. The aim of the first part of this work was to study the effect of the abundance of the spliceosomal snRNA components on splicing. Cells with a low abundance of the U12-type spliceosome were found to inefficiently process U12-type introns encoded by a transfected construct, but the expression levels of endogenous genes were not found to be affected by the abundance of the U12-type spliceosome. However, significant levels of endogenous unspliced U12-type intron-containing pre-mRNAs were detected in cells. Together these results support the idea that U12-type splicing may limit gene expression in some situations. The inefficiency of U12-type splicing has also promoted the idea that the U12-type spliceosome may control gene expression, limiting the mRNA levels of some U12-type intron-containing genes. While the identities of the primary target genes that contain U12-type introns are relatively well known, little has previously been known about the downstream genes and pathways potentially affected by the efficiency of U12-type intron processing. Here, the effects of U12-type splicing efficiency on a whole organism were studied in a Drosophila line with a mutation in an essential U12-type spliceosome component. Genes containing U12-type introns showed variable gene-specific responses to the splicing defect, which points to variation in the susceptibility of different genes to changes in splicing efficiency. Surprisingly, microarray screening revealed that metabolic genes were enriched among downstream effects, and that the phenotype could largely be attributed to one U12-type intron-containing mitochondrial gene. Gene expression control by the U12-type spliceosome could thus have widespread effects on metabolic functions in the organism. The subcellular localization of the U12-type spliceosome components was studied as a response to a recent dispute on the localization of the U12-type spliceosome. All components studied were found to be nuclear indicating that the processing of U12-type introns occurs within the nucleus, thus clarifying a question central to the field. The results suggest that the U12-type spliceosome can limit the expression of genes that contain U12-type introns in a gene-specific manner. Through its limiting role in pre-mRNA processing, the U12-type splicing activity can affect specific genetic pathways, which in the case of Drosophila are involved in metabolic functions.Aitotumallisissa eliöissä, joihin kuuluvat mm. kaikki monisoluiset eläimet ja kasvit, useimmat geenit ovat epäjatkuvia: koodaavat alueet vuorottelevat niissä ei-koodaavien jaksojen eli intronien kanssa. Geeni luetaan kokonaisuudessaan lähetti-RNA:ksi, minkä jälkeen intronit on poistettava toimivan lähetin tuottamiseksi. Intronien poiston eli silmukoinnin suorittaa moniosainen molekyylikoneisto, spliseosomi. Useimmilla monisoluisilla eliöillä on kaksi erilaista silmukointikoneistoa: U2-tyypin spliseosomi poistaa useimmat intronit, kun taas U12-tyypin spliseosomilla on tietyt kohdeintronit, jotka se löytää erityisten tunnistusjaksojen perusteella. Väitöskirjatyössäni olen tutkinut U12-tyypin spliseosomin erityispiirteitä, jotka eroavat yleisestä U2-tyypin silmukointikoneistosta ja tekevät U12-spliseosomista mahdollisen geeninsäätelymekanismin. Muutokset geeninsäätelyssä ovat esimerkiksi solujen erilaistumisen ja ympäristöön reagoimisen perusta, mikä tekee säätelymekanismeista keskeisen tutkimuskohteen molekyylibiologiassa. U12-tyypin intronien silmukoinnin on aiemmin havaittu olevan hitaampaa kuin tavallisen U2-tyypin, mikä viittaa siihen, että U12-tyypin introni voisi rajoittaa geenin toimintaa muodostamalla pullonkaulan lähetti-RNA:n muokkauksessa. U12-tyypin introneja esiintyy lähinnä geeneissä, joiden tehtävät liittyvät geneettisen informaation säilyttämiseen ja toimintaan soluissa, kuten DNA:n kopiointiin, geenien säätelyyn ja luentaan sekä solujenväliseen viestintään. On arveltu, että U12-tyypin silmukointi voi toimia tällaisten geenien säätelykeinona. Väitöskirjatyössäni havaittiin, että soluissa on normaalisti runsaasti lähetti-RNA:ita, joissa on silmukoimattomia U12-tyypin introneita, mikä viittaa siihen, että U12-tyypin silmukointi todella rajoittaa geenien ilmentymistä. Lisäksi todettiin, että U12-tyypin spliseosomin komponenttien määrä voi olla silmukointia rajoittava tekijä ainakin joissakin olosuhteissa. U12-tyypin intronin sisältävät geenit tunnetaan monissa eliöissä, mutta ei tiedetä, minkä geenien toimintaan ne puolestaan vaikuttavat. Tässä työssä U12-tyypin spliseosomin toiminnan estämisen vaikutuksia tutkittiin banaanikärpäsessä. Banaanikärpäsellä on vähemmän U12-tyypin introneja kuin esimerkiksi ihmisellä, mutta ihmisellä on U12-tyypin introni lähes kaikissa samoissa geeneissä kuin kärpäselläkin, mikä viittaa siihen, että U12-tyypin introneilla on tärkeä merkitys, jonka vuoksi ne säilyvät evoluutiossa. Tutkimuksissa havaittiin, että U12-tyypin silmukoinnin estäminen vaikutti ennen kaikkea perusaineenvaihduntaan sekä mitokondrioiden, solujen energiatehtaiden, toimintaan liittyviin geeneihin. Täten U12-tyypin spliseosomin aktiivisuuden taso voi vaikuttaa aivan eri tyyppisten geenien toimintaan kuin aiemmin on ajateltu. Kahden erillisen spliseosomikoneiston olemassaoloa on aiemmin pyritty selittämään myös ehdottamalla, että U12-tyypin spliseosomi toimisi solun tuman sijasta tuman ulkopuolella solulimassa. Tässä työssä selvitettiin U12-tyypin spliseosomin osien sijaintia solussa ja todettiin niiden sijaitsevan lähes yksinomaan tumassa. Tulos sopii hyvin yhteen sen kanssa, mitä ennestään tiedetään silmukoinnin yhteydestä muihin lähetti-RNA:ta muokkaaviin molekyylikoneistoihin, jotka myös toimivat tuman sisällä. Nämä ja muut viimeaikaiset tulokset viittaavat siihen, että syy toisen erillisen silmukointikoneiston ylläpitämiseen ei ole erilainen solunsisäinen sijainti vaan U12-tyypin spliseosomin rooli geeninsäätelyssä

Identification of transcriptional and metabolic programs related to mammalian cell size

SummaryBackgroundRegulation of cell size requires coordination of growth and proliferation. Conditional loss of cyclin-dependent kinase 1 in mice permits hepatocyte growth without cell division, allowing us to study cell size in vivo using transcriptomics and metabolomics.ResultsLarger cells displayed increased expression of cytoskeletal genes but unexpectedly repressed expression of many genes involved in mitochondrial functions. This effect appears to be cell autonomous because cultured Drosophila cells induced to increase cell size displayed a similar gene-expression pattern. Larger hepatocytes also displayed a reduction in the expression of lipogenic transcription factors, especially sterol-regulatory element binding proteins. Inhibition of mitochondrial functions and lipid biosynthesis, which is dependent on mitochondrial metabolism, increased the cell size with reciprocal effects on cell proliferation in several cell lines.ConclusionsWe uncover that large cell-size increase is accompanied by downregulation of mitochondrial gene expression, similar to that observed in diabetic individuals. Mitochondrial metabolism and lipid synthesis are used to couple cell size and cell proliferation. This regulatory mechanism may provide a possible mechanism for sensing metazoan cell size

Gene Expression Profiling of U12-Type Spliceosome Mutant Drosophila Reveals Widespread Changes in Metabolic Pathways : PLoS ONE

Background The U12-type spliceosome is responsible for the removal of a subset of introns from eukaryotic mRNAs. U12-type introns are spliced less efficiently than normal U2-type introns, which suggests a rate-limiting role in gene expression. The Drosophila genome contains about 20 U12-type introns, many of them in essential genes, and the U12-type spliceosome has previously been shown to be essential in the fly Methodology/Principal Findings We have used a Drosophila line with a P-element insertion in U6atac snRNA, an essential component of the U12-type spliceosome, to investigate the impact of U12-type introns on gene expression at the organismal level during fly development. This line exhibits progressive accumulation of unspliced U12-type introns during larval development and the death of larvae at the third instar stage. Surprisingly, microarray and RT-PCR analyses revealed that most genes containing U12-type introns showed only mild perturbations in the splicing of U12-type introns. In contrast, we detected widespread downstream effects on genes that do not contain U12-type introns, with genes related to various metabolic pathways constituting the largest group. Conclusions/Significance U12-type intron-containing genes exhibited variable gene-specific responses to the splicing defect, with some genes showing up- or downregulation, while most did not change significantly. The observed residual U12-type splicing activity could be explained with the mutant U6atac allele having a low level of catalytic activity. Detailed analysis of all genes suggested that a defect in the splicing of the U12-type intron of the mitochondrial prohibitin gene may be the primary cause of the various downstream effects detected in the microarray analysis.Peer reviewe

Requirement of TFIIH kinase subunit Mat1 for RNA Pol II C-terminal domain Ser5 phosphorylation, transcription and mRNA turnover

The relevance of serine 5 phosphorylation of RNA polymerase II carboxy-terminal domain during initiation has been difficult to determine in mammalian cells as no general in vivo Ser5 kinase has been identified. Here, we demonstrate that deletion of the TFIIH kinase subunit Mat1 in mouse fibroblasts leads to dramatically reduced Pol II Ser5 phosphorylation. This is associated with defective capping and reduced Ser2 phosphorylation, decreased Pol II progression into elongation and severely attenuated transcription detected through analysis of nascent mRNAs, establishing a general requirement for mammalian Mat1 in transcription. Surprisingly, the general defect in Pol II transcription in Mat1−/− fibroblasts is not reflected in the majority of steady-state mRNAs. This indicates widespread stabilization of mRNAs and points to the existence of a regulatory mechanism to stabilize mRNAs following transcriptional attenuation, thus revealing a potential caveat in similar studies limited to analysis of steady-state mRNAs

The abundance of the spliceosomal snRNPs is not limiting the splicing of U12-type introns

The rate of excision of U12-type introns has been reported to be slower than that of U2-type introns, suggesting a rate-limiting bottleneck that could down-regulate genes containing U12-type introns. The mechanistic reasons for this slower rate of intron excision are not known, but lower abundance of the U12-type snRNPs and slower rate of assembly or catalytic activity have been suggested. To investigate snRNP abundance we concentrated on the U4atac snRNA, which is the least abundant of the U12-type snRNAs and is limiting the formation of U4atac/U6atac complex. We identified mouse NIH-3T3 cell line isolates in which the level of both U4atac snRNA and U4atac/U6atac complexes is reduced to 10%–20% of the normal level. We used these cell lines to investigate splicing efficiency by transient transfection of a reporter gene containing a U12-type intron and by quantitative PCR analysis of endogenous genes. The splicing of the reporter U12-type intron was very inefficient, but the activity could be restored by overexpression of U4atac snRNA. Using these U4atac-deficient NIH-3T3 cells, we confirmed the results of previous studies showing that U12-type introns of endogenous genes are, indeed, excised more slowly than U2-type introns, but we found that the rate did not differ from that measured in cells displaying normal levels of U4atac snRNA. Thus our results suggest that the cellular abundance of the snRNPs does not limit U12-type intron splicing under normal conditions

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