snRNA Genes Research Articles

Finding the unexpected--how we identified a second class of introns and the U12-dependent spliceosome.

Finding the unexpected--how we identified a second class of introns and the U12-dependent spliceosome.

MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex.

Regulation of transcription elongation by RNA polymerase II (Pol II) is a key regulatory step in gene transcription. Recently, the little elongation complex (LEC)-which contains the transcription elongation factor ELL/EAF-was found to be required for the transcription of Pol II-dependent small nuclear RNA (snRNA) genes. Here we show that the human Mediator subunit MED26 plays a role in the recruitment of LEC to a subset of snRNA genes through direct interaction of EAF and the N-terminal domain (NTD) of MED26. Loss of MED26 in cells decreases the occupancy of LEC at a subset of snRNA genes and results in a reduction in their transcription. Our results suggest that the MED26-NTD functions as a molecular switch in the exchange of TBP-associated factor 7 (TAF7) for LEC to facilitate the transition from initiation to elongation during transcription of a subset of snRNA genes.

The Human Nuclear Exosome Targeting Complex Is Loaded onto Newly Synthesized RNA to Direct Early Ribonucleolysis

The RNA exosome complex constitutes the major nuclear eukaryotic 3'-5' exonuclease. Outside of nucleoli, the human nucleoplasmic exosome is directed to some of its substrates by the nuclear exosome targeting (NEXT) complex. How NEXT targets RNA has remained elusive. Using an in vivo crosslinking approach, we report global RNA binding sites of RBM7, a key component of NEXT. RBM7 associates broadly with RNA polymerase II-derived RNA, including pre-mRNA and short-lived exosome substrates such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and 3'-extended products from snRNA and replication-dependent histone genes. Within pre-mRNA, RBM7 accumulates at the 3' ends of introns, and pulse-labeling experiments demonstrate that RBM7/NEXT defines an early exosome-targeting pathway for 3'-extended snoRNAs derived from such introns. We propose that RBM7 is generally loaded onto newly synthesized RNA to accommodate exosome action in case of available unprotected RNA 3' ends.

The Myb domain of the largest subunit of SNAPc adopts different architectural configurations on U1 and U6 snRNA gene promoter sequences.

The small nuclear RNA (snRNA) activating protein complex (SNAPc) is essential for transcription of genes that encode the snRNAs. Drosophila melanogaster SNAPc (DmSNAPc) consists of three subunits (DmSNAP190, DmSNAP50 and DmSNAP43) that form a stable complex that recognizes an snRNA gene promoter element called the PSEA. Although all three subunits are required for sequence-specific DNA binding activity, only DmSNAP190 possesses a canonical DNA binding domain consisting of 4.5 tandem Myb repeats homologous to the Myb repeats in the DNA binding domain of the Myb oncoprotein. In this study, we use site-specific protein–DNA photo-cross-linking technology followed by site-specific protein cleavage to map domains of DmSNAP190 that interact with specific phosphate positions in the U6 PSEA. The results indicate that at least two DmSNAP190 Myb repeats contact the DNA in a significantly different manner when DmSNAPc binds to a U6 PSEA versus a U1 PSEA, even though the two PSEA sequences differ at only 5 of 21 nucleotide positions. The results are consistent with a model in which the specific DNA sequences of the U1 and U6 PSEAs differentially alter the conformation of DmSNAPc, leading to the subsequent recruitment of different RNA polymerases to the U1 and U6 gene promoters.

U1 snDNA clusters in grasshoppers: chromosomal dynamics and genomic organization.

The spliceosome, constituted by a protein set associated with small nuclear RNA (snRNA), is responsible for mRNA maturation through intron removal. Among snRNA genes, U1 is generally a conserved repetitive sequence. To unveil the chromosomal/genomic dynamics of this multigene family in grasshoppers, we mapped U1 genes by fluorescence in situ hybridization in 70 species belonging to the families Proscopiidae, Pyrgomorphidae, Ommexechidae, Romaleidae and Acrididae. Evident clusters were observed in all species, indicating that, at least, some U1 repeats are tandemly arrayed. High conservation was observed in the first four families, with most species carrying a single U1 cluster, frequently located in the third or fourth longest autosome. By contrast, extensive variation was observed among Acrididae, from a single chromosome pair carrying U1 to all chromosome pairs carrying it, with occasional occurrence of two or more clusters in the same chromosome. DNA sequence analysis in Eyprepocnemis plorans (species carrying U1 clusters on seven different chromosome pairs) and Locusta migratoria (carrying U1 in a single chromosome pair) supported the coexistence of functional and pseudogenic lineages. One of these pseudogenic lineages was truncated in the same nucleotide position in both species, suggesting that it was present in a common ancestor to both species. At least in E. plorans, this U1 snDNA pseudogenic lineage was associated with 5S rDNA and short interspersed elements (SINE)-like mobile elements. Given that we conclude in grasshoppers that the U1 snDNA had evolved under the birth-and-death model and that its intragenomic spread might be related with mobile elements.

Conditional inactivation of Upstream Binding Factor reveals its epigenetic functions and the existence of a somatic nucleolar precursor body.

Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me3, canonical histone H1 and HP1α, but not by de novo DNA methylation. Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs).

DSIF and NELF interact with Integrator to specify the correct post-transcriptional fate of snRNA genes.

The elongation factors DSIF and NELF are responsible for promoter-proximal RNA polymerase II (Pol II) pausing. NELF is also involved in 3' processing of replication-dependent histone genes, which produce non-polyadenylated mRNAs. Here we show that DSIF and NELF contribute to the synthesis of small nuclear RNAs (snRNAs) through their association with Integrator, the large multisubunit complex responsible for 3' processing of pre-snRNAs. In HeLa cells, Pol II, Integrator, DSIF and NELF accumulate at the 3' end of the U1 snRNA gene. Knockdown of NELF results in misprocessing of U1, U2, U4 and U5 snRNAs, while DSIF is required for proper transcription of these genes. Knocking down NELF also disrupts transcription termination and induces the production of polyadenylated U1 transcripts caused by an enhanced recruitment of cleavage stimulation factor. Our results indicate that NELF plays a key role in determining the post-transcriptional fate of Pol II-transcribed genes.

Comparative Chromosome Mapping of U2 snRNA and 5S rRNA Genes in Gymnotus Species (Gymnotiformes, Gymnotidae): Evolutionary Dynamics and Sex Chromosome Linkage in G. pantanal

A comparative mapping of U2 small nuclear RNA (snRNA) and 5S ribosomal RNA (rRNA) genes was performed in 6 Gymnotus species. All species analyzed presented the U2 snDNA organized in conspicuous blocks and not co-located with rRNA genes. In addition, 5 species showed the U2 snDNA located in a single pair of chromosomes, which seems to be a conserved trait in this genus. Conversely, G. pantanal was the only species displaying several terminal signals in different chromosome pairs, including the X₁ sex chromosome but not the Y chromosome. This is the first report of U2 snRNA genes in sex chromosomes of fishes. The absence of sites in the Y chromosome of G. pantanal indicates a possible loss of terminal segments of the chromosomes involved in the Y formation.

Human snRNA genes use polyadenylation factors to promote efficient transcription termination

RNA polymerase II transcribes both protein coding and non-coding RNA genes and, in yeast, different mechanisms terminate transcription of the two gene types. Transcription termination of mRNA genes is intricately coupled to cleavage and polyadenylation, whereas transcription of small nucleolar (sno)/small nuclear (sn)RNA genes is terminated by the RNA-binding proteins Nrd1, Nab3 and Sen1. The existence of an Nrd1-like pathway in humans has not yet been demonstrated. Using the U1 and U2 genes as models, we show that human snRNA genes are more similar to mRNA genes than yeast snRNA genes with respect to termination. The Integrator complex substitutes for the mRNA cleavage and polyadenylation specificity factor complex to promote cleavage and couple snRNA 3′-end processing with termination. Moreover, members of the associated with Pta1 (APT) and cleavage factor I/II complexes function as transcription terminators for human snRNA genes with little, if any, role in snRNA 3′-end processing. The gene-specific factor, proximal sequence element-binding transcription factor (PTF), helps clear the U1 and U2 genes of nucleosomes, which provides an easy passage for pol II, and the negative elongation factor facilitates termination at the end of the genes where nucleosome levels increase. Thus, human snRNA genes may use chromatin structure as an additional mechanism to promote efficient transcription termination in vivo.

Differential Utilization of TATA Box-binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459-469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

The Little Elongation Complex Functions at Initiation and Elongation Phases of snRNA Gene Transcription

The small nuclear RNA (snRNA) genes have been widely used as a model system for understanding transcriptional regulation due to the unique aspects of their promoter structure, selectivity for either RNA polymerase (Pol) II or III, and because of their unique mechanism of termination that is tightly linked with the promoter. Recently, we identified the little elongation complex (LEC) in Drosophila that is required for the expression of Pol II-transcribed snRNA genes. Here, using Drosophila and mammalian systems, we provide genetic and molecular evidence that LEC functions in at least two phases of snRNA transcription: an initiation step requiring the ICE1 subunit, and an elongation step requiring ELL.

Draft Assembly of the Symbiodinium minutum Nuclear Genome Reveals Dinoflagellate Gene Structure

Dinoflagellates are known for their capacity to form harmful blooms (e.g., "red tides") and as symbiotic, photosynthetic partners for corals. These unicellular eukaryotes have permanently condensed, liquid-crystalline chromosomes and immense nuclear genome sizes, often several times the size of the human genome. Here we describe the first draft assembly of a dinoflagellate nuclear genome, providing insights into its genome organization and gene inventory. Sequencing reads from Symbiodinium minutum were assembled into 616 Mbp gene-rich DNA regions that represented roughly half of the estimated 1,500 Mbp genome of this species. The assembly encoded ∼42,000 protein-coding genes, consistent with previous dinoflagellate gene number estimates using transcriptomic data. The Symbiodinium genome contains duplicated genes for regulator of chromosome condensation proteins, nearly one-third of which have eukaryotic orthologs, whereas the remainder have most likely been acquired through bacterial horizontal gene transfers. Symbiodinium genes are enriched in spliceosomal introns (mean= 18.6 introns/gene). Donor and acceptor splice sites are unique, with 5' sites utilizing not only GT but also GC and GA, whereas at 3' sites, a conserved G is present after AG. All spliceosomal snRNA genes (U1-U6) are clustered in the genome. Surprisingly, the Symbiodinium genome displays unidirectionally aligned genes throughout the genome, forming a cluster-like gene arrangement. We show here that a dinoflagellate genome exhibits unique and divergent characteristics when compared to those of other eukaryotes. Our data elucidate the organization and gene inventory of dinoflagellates and lay the foundation for future studies of this remarkable group of eukaryotes.

A Superfamily of DNA Transposons Targeting Multicopy Small RNA Genes

Target-specific integration of transposable elements for multicopy genes, such as ribosomal RNA and small nuclear RNA (snRNA) genes, is of great interest because of the relatively harmless nature, stable inheritance and possible application for targeted gene delivery of target-specific transposable elements. To date, such strict target specificity has been observed only among non-LTR retrotransposons. We here report a new superfamily of sequence-specific DNA transposons, designated Dada. Dada encodes a DDE-type transposase that shows a distant similarity to transposases encoded by eukaryotic MuDR, hAT, P and Kolobok transposons, as well as the prokaryotic IS256 insertion element. Dada generates 6–7 bp target site duplications upon insertion. One family of Dada DNA transposons targets a specific site inside the U6 snRNA genes and are found in various fish species, water flea, oyster and polycheate worm. Other target sequences of the Dada transposons are U1 snRNA genes and different tRNA genes. The targets are well conserved in multicopy genes, indicating that copy number and sequence conservation are the primary constraints on the target choice of Dada transposons. Dada also opens a new frontier for target-specific gene delivery application.

Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading.

Supernumerary chromosomes (B chromosomes) occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C0t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes.

Kinetic Competition between Elongation Rate and Binding of NELF Controls Promoter-Proximal Pausing

Pausing of RNA polymerase II (Pol II) 20-60 bp downstream of transcription start sites is a major checkpoint during transcription in animal cells. Mechanisms that control pausing are largely unknown. We developed permanganate-ChIP-seq to evaluate the state of Pol II at promoters throughout the Drosophila genome, and a biochemical system that reconstitutes promoter-proximal pausing to define pausing mechanisms. Stable open complexes of Pol II are largely absent from the transcription start sites of most mRNA genes but are present at snRNA genes and the highly transcribed heat shock genes following their induction. The location of the pause is influenced by the timing between when NELF loads onto Pol II and how fast Pol II escapes the promoter region. Our biochemical analysis reveals that the sequence-specific transcription factor, GAF, orchestrates efficient pausing by recruiting NELF to promoters before transcription initiation and by assisting in loading NELF onto Pol II after initiation.

Differential contacts of the 4.5 Myb repeats of SNAP190 with the DNA of U1 and U6 snRNA gene promoters

U1 and U6 snRNA genes are transcribed by RNA polymerases II and III respectively, yet the transcription of each is dependent upon the small nuclear RNA activating protein complex (SNAPc). In D. melanogaster, DmSNAPc recognizes a 21 base‐pair DNA sequence, the PSEA, located approximately 40–60 base pairs upstream of the transcription start site of each gene. DmSNAPc is a heterotrimer of subunits named DmSNAP190, DmSNAP50, and DmSNAP43. All three subunits are required for DNA binding activity, but only DmSNAP190 contains a “canonical” DNA binding domain that consists of 4.5 tandem Myb repeats. Myb repeats are each ~50 amino acid residues in length and were first identified as forming the DNA binding domain of the Myb oncoprotein. By using a site‐specific protein‐DNA photo‐cross‐linking assay combined with site‐specific protein digestion, we have been able to map where each of the 4.5 Myb repeats of DmSNAP190 contact the DNA of the U1 and U6 PSEAs. Although many of the contact points are similar, the Myb repeats of DmSNAP190 interact with the U1 DNA primarily through the major groove, but new and distinct Myb repeats‐DNA contacts that span the minor groove are observed when DmSNAPc binds to a U6 PSEA. This conformational shift in DmSNAP190 binding is likely important in determining the RNA polymerase specificity of the U1 and U6 gene promoters. (Supported by NSF and in part by the California Metabolic Research Foundation.)

Single or Multiple Gene Silencing Directed by U6 Promoter-Based shRNA Vectors Facilitates Efficient Functional Genome Analysis in Medicago truncatula

Short hairpin RNA (shRNA) expression vectors are an effective means to deliver double-stranded RNA that mediates sequence-specific cleavage of target transcripts in RNA interference (RNAi). Previously, we constructed shRNA expression vectors based on U6 small nuclear RNA (snRNA) gene promoters of Medicago truncatula. To facilitate screening of shRNA candidates, a protoplast-based dual luciferase assay system was developed, which consists of a shRNA-expressing effector plasmid and a target gene-carrying reporter plasmid. RNAi efficiency of a given shRNA could be measured by comparing two luciferase activities derived from transcripts with an intact or interrupted 3′ untranslated region as a result of RNAi. Using a multiple U6-shRNA cassette plasmid that enables targeting of multiple genes simultaneously, the expression of two Δ1-pyrroline-5-carboxylate synthetase genes (MtP5CS1 and MtP5CS2) was silenced with a U6 plasmid carrying two 27-nucleotide-stem shRNA cassettes targeting each gene. The decreased transcript levels were accompanied by decreased protein levels and decreased free proline contents. To examine possible U6-shRNA-mediated RNAi in nodules, two remorin-encoding MtREM genes were targeted by single and double U6-shRNA plasmids. Silencing of MtREM2.2, an essential nodulation gene, MtREM1.1, an uncharacterized gene, or both inhibited nodule formation in transgenic roots. Moreover, M. truncatula roots transformed with a suppressor-overexpresser construct wherein MtREM2.2-targeting U6-shRNA and GmREM2.3-overexpressing cassettes were placed tandemly showed decreased MtREM2.2 transcripts yet survived nitrogen-free medium, indicating complementation of MtREM2.2 with the overexpressed soybean gene. These results demonstrate that U6 promoter-based shRNA-expressing plasmids are valuable for multiple gene functional analyses in protoplasts and nodules of M. truncatula.

Coilin Displays Differential Affinity for Specific RNAs In Vivo and Is Linked to Telomerase RNA Biogenesis

Coilin is widely known as the protein marker of the Cajal body, a subnuclear domain important to the biogenesis of small nuclear ribonucleoproteins and telomerase, complexes that are crucial to pre-messenger RNA splicing and telomere maintenance, respectively. Extensive studies have characterized the interaction between coilin and the various other protein components of CBs and related subnuclear domains; however, only a few have examined interactions between coilin and nucleic acid. We have recently published that coilin is tightly associated with nucleic acid, displays RNase activity in vitro, and is redistributed to the ribosomal RNA (rRNA)-rich nucleoli in cells treated with the DNA-damaging agents cisplatin and etoposide. Here, we report a specific in vivo association between coilin and rRNA, U small nuclear RNA (snRNA), and human telomerase RNA, which is altered upon treatment with DNA-damaging agents. Using chromatin immunoprecipitation, we provide evidence of coilin interaction with specific regions of U snRNA gene loci. We have also utilized bacterially expressed coilin fragments in order to map the region(s) important for RNA binding and RNase activity in vitro. Additionally, we provide evidence of coilin involvement in the processing of human telomerase RNA both in vitro and in vivo.

Isolation and Characterization of Medicago truncatula U6 Promoters for the Construction of Small Hairpin RNA-Mediated Gene Silencing Vectors

RNA silencing using vector-based double-stranded RNA is an attractive strategy to suppress gene expression in plants. Especially, a short hairpin RNA (shRNA)-mediated approach is potent for specific target gene silencing. Here, shRNA expression vectors were constructed based on U6 small nuclear RNA (snRNA) gene promoters in Medicago truncatula. Ten U6 snRNA gene loci identified showed highly homologous sequences with those of other eukaryotes. Their RNA polymerase III-specific promoters had a conserved upstream sequence element and TATA box that were located approximately three helical DNA turns apart. The U6 promoters were also insensitive to α-amanitin, actively guiding transcription of fused GUS reporter gene fragments. When the U6 fusion constructs were introduced into M. truncatula via Agrobacterium rhizogenes-mediated transformation, GUS fragment transcripts were detected in nearly every transformed root albeit in varying amounts. Consistently, under the control of U6 promoters, constructs of 21-nucleotide-stem shRNAs directed silencing of GUS expression in transformed roots. Moreover, a phytoene desaturase gene (MtPDS3)-targeting 27-nucleotide-stem U6-shRNA constructs, when introduced into hairy roots, decreased the MtPDS3 transcript levels by approximately 80 % as a result of endogenous gene silencing, as verified by the presence of shPDS-derived siRNAs detected by stem-loop PCR. The U6 promoter-directed shRNA expression plasmids constructed herein can be a useful tool for functional gene analyses in this model legume.

Genomic Study of RNA Polymerase II and III SNAPc-Bound Promoters Reveals a Gene Transcribed by Both Enzymes and a Broad Use of Common Activators

SNAPc is one of a few basal transcription factors used by both RNA polymerase (pol) II and pol III. To define the set of active SNAPc-dependent promoters in human cells, we have localized genome-wide four SNAPc subunits, GTF2B (TFIIB), BRF2, pol II, and pol III. Among some seventy loci occupied by SNAPc and other factors, including pol II snRNA genes, pol III genes with type 3 promoters, and a few un-annotated loci, most are primarily occupied by either pol II and GTF2B, or pol III and BRF2. A notable exception is the RPPH1 gene, which is occupied by significant amounts of both polymerases. We show that the large majority of SNAPc-dependent promoters recruit POU2F1 and/or ZNF143 on their enhancer region, and a subset also recruits GABP, a factor newly implicated in SNAPc-dependent transcription. These activators associate with pol II and III promoters in G1 slightly before the polymerase, and ZNF143 is required for efficient transcription initiation complex assembly. The results characterize a set of genes with unique properties and establish that polymerase specificity is not absolute in vivo.