Human Small Nuclear RNA Research Articles

Regulation of Human RNA Polymerase III Transcription by DNMT1 and DNMT3a DNA Methyltransferases

The human small nuclear RNA (snRNA) and small cytoplasmic RNA (scRNA) gene families encode diverse non-coding RNAs that influence cellular growth and division. Many snRNA and scRNA genes are related via their compact and yet powerful promoters that support RNA polymerase III transcription. We have utilized the human U6 snRNA gene family to examine the mechanism for regulated transcription of these potent transcription units. Analysis of nine U6 family members showed enriched CpG density within the promoters of actively transcribed loci relative to inert genes, implying a relationship between gene potency and DNA methylation. Indeed, both pharmacological inhibition of DNA methyltransferase (DNMT) activity and the forced diminution of DNMT-1, DNMT-3a, and DNMT-3b by siRNA targeting resulted in increased U6 levels in asynchronously growing MCF7 adenocarcinoma cells. In vitro transcription assays further showed that template methylation impedes U6 transcription by RNA polymerase III. Both DNMT-1 and DNMT-3a were detected at the U6-1 locus by chromatin immunoprecipitation directly linking these factors to RNA polymerase III regulation. Despite this association, the endogenous U6-1 locus was not substantially methylated in actively growing cells. However, both DNMT occupancy and low frequency methylation were correlated with increased Retinoblastoma tumor suppressor (RB) expression, suggesting that the RB status can influence specific epigenetic marks.

Role of the HIV-1 Positive Elongation Factor P-TEFb and Inhibitors Thereof

Transcription is considered to be a crucial step in the replication cycle of HIV-1. Tat regulates an early step of transcription elongation. The positive elongation factor P-TEFb, a heterodimer containing a catalytic subunit (CDK9) and unique regulatory cyclins (CycT1), is required for HIV-1 Tat transcriptional activation. This is a potential target for new HIV-1 transcription inhibitors. Without P-TEFb, transactivation is restrained and only short transcripts are generated. All the P-TEFb inhibitors can suppress the HIV-1 transactivation process by inhibition of CycT1, CDK9 or their interaction. Several low-molecular-weight compounds such as flavopiridol, roscovitine and the human small nuclear RNA 7SK which have been showed to possess potent anti-HIV activity by interfering with P-TEFb functions are reviewed in this article.

Transcriptional regulation of human small nuclear RNA genes.

The products of human snRNA genes have been frequently described as performing housekeeping functions and their synthesis refractory to regulation. However, recent studies have emphasized that snRNA and other related non-coding RNA molecules control multiple facets of the central dogma, and their regulated expression is critical to cellular homeostasis during normal growth and in response to stress. Human snRNA genes contain compact and yet powerful promoters that are recognized by increasingly well-characterized transcription factors, thus providing a premier model system to study gene regulation. This review summarizes many recent advances deciphering the mechanism by which the transcription of human snRNA and related genes are regulated.

The Protein Kinase CK2 Phosphorylates SNAP190 to Negatively Regulate SNAPC DNA Binding and Human U6 Transcription by RNA Polymerase III

Human U6 small nuclear RNA gene transcription by RNA polymerase III requires the general transcription factor SNAP(C), which binds to human small nuclear RNA core promoter elements and nucleates pre-initiation complex assembly with the Brf2-TFIIIB complex. Multiple components in this pathway are phosphorylated by the protein kinase CK2, including the Bdp1 subunit of the Brf2-TFIIIB complex, and RNA polymerase III, with negative and positive outcomes for U6 transcription, respectively. However, a role for CK2 phosphorylation of SNAP(C) in U6 transcription has not been defined. In this report, we investigated the role of CK2 in modulating the transcriptional properties of SNAP(C) and demonstrate that within SNAP(C), CK2 phosphorylates the N-terminal half of the SNAP190 subunit at two regions (amino acids 20-63 and 514-545) that each contain multiple CK2 consensus sites. SNAP190 phosphorylation by CK2 inhibits both SNAP(C) DNA binding and U6 transcription activity. Mutational analyses of SNAP190 support a model wherein CK2 phosphorylation triggers an allosteric inhibition of the SNAP190 Myb DNA binding domain.

Study of Claudin Function by RNA Interference

Claudins are tight junction proteins that play a key selectivity role in the paracellular conductance of ions. Numerous studies of claudin function have been carried out using the overexpression strategy to add new claudin channels to an existing paracellular protein background. Here, we report the systematic knockdown of endogenous claudin gene expression in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells using small interfering RNA against claudins 1-4 and 7. In MDCK cells (showing cation selectivity), claudins 2, 4, and 7 are powerful effectors of paracellular Na+ permeation. Removal of claudin-2 depressed the permeation of Na+ and resulted in the loss of cation selectivity. Loss of claudin-4 or -7 expression elevated the permeation of Na+ and enhanced the proclivity of the tight junction for cations. On the other hand, LLC-PK1 cells express little endogenous claudin-2 and show anion selectivity. In LLC-PK1 cells, claudin-4 and -7 are powerful effectors of paracellular Cl- permeation. Knockdown of claudin-4 or -7 expression depressed the permeation of Cl- and caused the tight junction to lose the anion selectivity. In conclusion, claudin-2 functions as a paracellular channel to Na+ to increase the cation selectivity of the tight junction; claudin-4 and -7 function either as paracellular barriers to Na+ or as paracellular channels to Cl-, depending upon the cellular background, to decrease the cation selectivity of the tight junction.

The Unorthodox SNAP50 Zinc Finger Domain Contributes to Cooperative Promoter Recognition by Human SNAPC

Human small nuclear RNA gene transcription by RNA polymerases II and III depends upon promoter recognition by the SNAPC general transcription factor. DNA binding by SNAPC involves direct DNA contacts by the SNAP190 subunit in cooperation with SNAP50 and SNAP43. The data presented herein shows that SNAP50 plays an important role in DNA binding by SNAPC through its zinc finger domain. The SNAP50 zinc finger domain contains 15 cysteine and histidine residues configured in two potential zinc coordination arrangements. Individual alanine substitution of each cysteine and histidine residue demonstrated that eight sites are important for DNA binding by SNAPC. However, metal binding studies revealed that SNAPC contains a single zinc atom indicating that only one coordination site functions as a zinc finger. Of the eight residues critical for DNA binding, four cysteine residues were also essential for both U1 and U6 transcription by RNA polymerase II and III, respectively. Surprisingly, the remaining four residues, although critical for U1 transcription could support partial U6 transcription. DNA binding studies showed that defects in DNA binding by SNAPC alone could be suppressed through cooperative DNA binding with another member of the RNA polymerase III general transcription machinery, TFIIIB. These results suggest that these eight cysteine and histidine residues perform different functions during DNA binding with those residues involved in zinc coordination likely performing a dominant role in domain stabilization and the others involved in DNA binding. These data further define the unorthodox SNAP50 zinc finger region as an evolutionarily conserved DNA binding domain.

A role for Yin Yang-1 (YY1) in the assembly of snRNA transcription complexes

The RNA polymerase (pol) II and III human small nuclear RNA (snRNA) genes have very similar promoters and recruit a number of common factors. In particular, both types of promoters utilize the small nuclear RNA activating protein complex (SNAP c) and the TATA box binding protein (TBP) for basal transcription, and are activated by Oct-1. We find that SNAP c purified from cell lines expressing tagged SNAP c subunits is associated with Yin Yang-1 (YY1), a factor implicated in both activation and repression of transcription. Recombinant YY1 accelerates the binding of SNAP c to the proximal sequence element, its target within snRNA promoters. Moreover, it enhances the formation of a complex on the pol III U6 snRNA promoter containing all the factors (SNAP c, TBP, TFIIB-related factor 2 (Brf2), and B double prime 1 (Bdp1)) that are sufficient to direct in vitro U6 transcription when complemented with purified pol III, as well as that of a subcomplex containing TBP, Brf2, and Bdp1. YY1 is found on both the RNA polymerase II U1 and the RNA polymerase III U6 promoters as determined by chromatin immunoprecipitations. Thus, YY1 represents a new factor that participates in transcription complexes formed on both pol II and III promoters.

The Trypanosoma brucei spliced leader RNA and rRNA gene promoters have interchangeable TbSNAP50-binding elements.

In the protist parasite Trypanosoma brucei, the small nuclear spliced leader (SL) RNA and the large rRNAs are key molecules for mRNA maturation and protein synthesis, respectively. The SL RNA gene (SLRNA) promoter recruits RNA polymerase II and consists of a bipartite upstream sequence element (USE) and an element close to the transcription initiation site. Here, we analyzed the distal part of the ribosomal (RRNA) promoter and identified two sequence blocks which, in reverse orientation, closely resemble the SLRNA USE by both sequence and spacing. A detailed mutational analysis revealed that the ribosomal (r)USE is essential for efficient RRNA transcription in vivo and that it functions in an orientation-dependent manner. Moreover, we showed that USE and rUSE are functionally interchangeable and that rUSE stably interacted with an essential factor of SLRNA transcription. Finally, we demonstrated that the T.brucei homolog of the recently characterized transcription factor p57 of the related organism Leptomonas seymouri specifically bound to USE and rUSE. Since p57 and its T.brucei counterpart are homologous to SNAP50, a component of the human small nuclear RNA gene activation protein complex (SNAPc), both SLRNA and RRNA transcription in T.brucei may depend on a SNAPc-like transcription factor.

Retroviral vectors designed for targeted expression of RNA polymerase III-driven transcripts: a comparative study

Retroviral gene delivery systems for RNA polymerase II (RNA pol II)-based promoters have been developed and are widely used in gene transfer studies. In contrast, gene delivery systems with RNA pol III-based expression cassettes have not been studied comprehensively, although therapeutic applications (e.g., ribozymes, antisense, triplex RNA and RNA decoys) have been proposed. In this report, we describe retroviral vectors designed to optimize expression of short chimeric RNAs transcribed from a number of RNA pol III promoters. Our results show that all analysed RNA pol III expression cassettes (tRNA, U6, Ad VA1), regardless of orientation, do not transcribe efficiently when located between the retroviral long terminal repeats (LTRs). In contrast, high steady-state expression levels can be achieved by inserting the RNA pol III expression cassette into the U3 region of the LTR (double-copy design). Compared to human tRNA gene promoters (tRNA Met, tRNA Val), the human small nuclear RNA U6 gene ( U6) and the adenovirus virus-associated RNA 1 (Ad VA1) gene promoters yielded higher expression levels. The majority of the chimeric U6-derived transcripts were detected in the nuclear RNA fraction, and the VA1 and tRNA-driven transcripts were predominantly detected in the cytoplasmic compartments. This report is the first comparative study of RNA pol III-driven promoters expressing short chimeric transcripts leading to an optimized retroviral-vector design.

Pre-messenger RNA Splicing of Transcripts Synthesized from Human Small Nuclear RNA Gene Promoters

In order to explore the coupling of transcription with splicing in mammalian cells we have prepared hybrid genes in which either the human U2 promoter, recognized by RNA polymerase II, or the human U6 promoter, recognized by RNA polymerase III, was fused to an intron-containing gene segment. Neither human small nuclear RNA gene contains an intron although U6 genes from some species of yeast contain a short intervening sequence. Following transfection of human cells and analysis of specific RNAs by primer extension we found that the chimeric U2 promoter-derived transcript was efficiently spliced but the RNA polymerase III transcript driven by the U6 promoter remained unspliced. Hence, the splicing apparatus differentiates between transcripts produced from two closely related promoters that are distinguished by RNA polymerase selectivity.

Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing complex activates transcription from snRNA promoters through the PSE.

In the human small nuclear RNA (snRNA) promoters, the presence of a TATA box recognized by the TATA box-binding protein (TBP) determines the selection of RNA polymerase III over RNA polymerase II. The RNA polymerase II snRNA promoters are, therefore, good candidates for TBP-independent promoters. We show here, however, that TBP activates transcription from RNA polymerase II snRNA promoters through a non-TATA box element, the snRNA proximal sequence element (PSE), as part of a new snRNA-activating protein complex (SNAPc). In contrast to the previously identified TBP-containing complexes SL1, TFIID, and TFIIIB, which appear dedicated to transcription by a single RNA polymerase, SNAPc is also essential for RNA polymerase III transcription from the U6 snRNA promoter. The U6 initiation complex appears to contain two forms of TBP, one bound to the TATA box and one bound to the PSE as a part of SNAPc, suggesting that multiple TBP molecules can have different functions within a single promoter.

Clustered genes for human U2 RNA.

Genes for the human small nuclear RNA U2 are present within 6.2-kilobase-pair-long tandem repeats. The haploid human genome contains approximately 20 such repeats, organized in one or a few very large clusters.

U1 small nuclear ribonucleoprotein studied by in vitro assembly.

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody-precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA-protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.

Pseudogenes for human small nuclear RNA U3 appear to arise by integration of self-primed reverse transcripts of the RNA into new chromosomal sites

We find that both human and rat U3 snRNA can function as self-priming templates for AMV reverse transcriptase in vitro. The 74 base cDNA is primed by the 3′ end of intact U3 snRNA, and spans the characteristically truncated 69 or 70 base U3 sequence found in four different human U3 pseudogenes. The ability of human and rat U3 snRNA to self-prime is consistent with a U3 secondary structure model derived by a comparison between rat U3 snRNA and the homologous D2 snRNA from Dictyostelium discoideum. We propose that U3 pseudogenes are generated in vivo by integration of a self-primed cDNA copy of U3 snRNA at new chromosomal sites. We also consider the possibility that the same cDNA mediates gene conversion at the 5′ end of bona fide U3 genes where, over the entire region spanned by the U3 cDNA, the two rat U3 sequence variants U3A and U3B are identical.