Small Nuclear RNA-activating Protein Complex Research Articles

The SNAPc complex mediates starvation-induced trans-splicing in Caenorhabditis elegans

Dietary restriction usually suppresses biosynthesis but activates catabolic pathways in animals. However, the short-term starvation enhances biosynthetic activities and promotes ribosomal biogenesis in adult Caenorhabditis elegans. The mechanism underlying the processes remains largely unknown. Here, we find that the short-term starvation enhances the SL1 trans-splicing of translation-related genes in adult C. elegans by transcriptome analysis. The small nuclear RNA-activating protein complex (SNAPc) promotes SL RNA production and mediates starvation-induced trans-splicing. TOFU-5, a core factor in the upstream sequence transcription complex (USTC) essential for piRNA production, is also involved in the starvation-induced trans-splicing processes. Knocking down components of the SNAPc complex and tofu-5 extends worm survival under starvation conditions. Taken together, our study highlights the importance of SL trans-splicing in the nutrition response and reveals a mechanism of the survival regulation by food deprivation via SNAPc and TOFU-5.

SNPC-1.3 is a sex-specific transcription factor that drives male piRNA expression in C. elegans.

Piwi-interacting RNAs (piRNAs) play essential roles in silencing repetitive elements to promote fertility in metazoans. Studies in worms, flies, and mammals reveal that piRNAs are expressed in a sex-specific manner. However, the mechanisms underlying this sex-specific regulation are unknown. Here we identify SNPC-1.3, a male germline-enriched variant of a conserved subunit of the small nuclear RNA-activating protein complex, as a male-specific piRNA transcription factor in Caenorhabditis elegans. SNPC-1.3 colocalizes with the core piRNA transcription factor, SNPC-4, in nuclear foci of the male germline. Binding of SNPC-1.3 at male piRNA loci drives spermatogenic piRNA transcription and requires SNPC-4. Loss of snpc-1.3 leads to depletion of male piRNAs and defects in male-dependent fertility. Furthermore, TRA-1, a master regulator of sex determination, binds to the snpc-1.3 promoter and represses its expression during oogenesis. Loss of TRA-1 targeting causes ectopic expression of snpc-1.3 and male piRNAs during oogenesis. Thus, sexually dimorphic regulation of snpc-1.3 expression coordinates male and female piRNA expression during germline development.

Assembly of SNAPc, Bdp1, and TBP on the U6 snRNA Gene Promoter in Drosophila melanogaster.

U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.

Proteomics analysis of proteins interacting with heat shock factor 1 in squamous cell carcinoma of the cervix.

Protein interactions are crucial for maintaining homeostasis. Heat shock factor 1 (HSF1), a transcription factor that interacts with various proteins, is highly expressed in squamous cell carcinoma (SCC) of the cervix. The aim of the present study was to investigate the protein interaction profile of HSF1 in cervical SCC. Proteins interacting with HSF1 in SCC tissue and non-cancerous control (Ctrl) tissue were obtained by immunoprecipitation, separated by SDS-PAGE, identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and analyzed using bioinformatics methods. A total of 220 and 241 proteins were identified by mass spectrometry in the tissues of Ctrl and SCC samples, respectively, among which 172 were detected exclusively in SCC (Pro-S), 151 exclusively in Ctrl (Pro-C) and 69 in both groups (Pro-B). The protein interaction profiles were different in each group; the STRING database identified three proteins that interacted with HSF1 directly, including insulin-like growth factor 1 receptor and small nuclear RNA-activating protein complex subunit 4 in Pro-C and small ubiquitin-related modifier 1 in Pro-S. Functional enrichment analysis of Gene Ontology revealed that the top terms were alternative splicing in Pro-S and polymorphism in Pro-C. In Pro-S, more categories were related to protein modification, such as phosphorylation, ubiquitination and acetylation. Therefore, HSF1 may influence the occurrence and development of cervical SCC by interacting with specific proteins.

Bdp1 interacts with SNAPc bound to a U6, but not U1, snRNA gene promoter element to establish a stable protein-DNA complex.

In metazoans, U6 small nuclear RNA (snRNA) gene promoters utilize a proximal sequence element (PSE) recognized by the small nuclear RNA-activating protein complex (SNAPc). SNAPc interacts with the transcription factor TFIIIB, which consists of the subunits TBP, Brf1 (Brf2 in vertebrates), and Bdp1. Here, we show that, in Drosophila melanogaster, DmSNAPc directly recruits Bdp1 to the U6 promoter, and we identify an 87-residue region of Bdp1 involved in this interaction. Importantly, Bdp1 recruitment requires that DmSNAPc be bound to a U6 PSE rather than a U1 PSE. This is consistent with the concept that DmSNAPc adopts different conformations on U6 and U1 PSEs, which lead to the subsequent recruitment of distinct general transcription factors and RNA polymerases for U6 and U1 gene transcription.

New Insights into the Assembly Mechanism of an RNA Polymerase III‐Specific Transcription Complex on a Drosophila U6 snRNA Gene Promoter

In higher eukaryotes, RNA polymerase III (Pol III) promoters at U6 snRNA genes consist of a TATA box, recognized by TFIIIB, and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). In the fruit fly Drosophila melanogaster, DmSNAPc consists of three subunits DmSNAP190, DmSNAP50, and DmSNAP43; likewise TFIIIB also consists of three subunits, most commonly TBP, Brf1 and Bdp1. At Drosophila tRNA and 5S RNA gene promoters, TBP‐related factor 1 (TRF1) is utilized in place of TBP, but at U6 promoters the canonical TBP is utilized for Pol III transcription (Verma et al. 2013, JBC 288, 27564–27570).Site‐specific protein‐DNA photo‐cross‐linking studies of DmSNAPc and TFIIIB to U6 promoter DNA indicated that Bdp1 is in close proximity to DmSNAP43 and DmSNAP190 on the U6 promoter (Kang et al. 2016, FEBS Lett 590, 1488–1497). This suggested that the interaction between DmSNAPc and TFIIIB may be mediated, at least in part, by Bdp1. We have investigated this further by electrophoretic mobility shift assays (EMSAs). Surprisingly, we found that DmSNAPc, when bound to the U6 PSE, can recruit Bdp1 to the DNA in the absence of TBP and Brf1. Furthermore, EMSAs indicated that the DmSNAPc‐Bdp1 complex, when bound to U6 promoter DNA, can recruit TBP to form a DmSNAPc‐Bdp1‐TBP‐DNA complex of increased stability.In order to understand the protein‐protein interactions taking place, truncation mutations of Bdp1 were used. It was discovered that an area between amino acid 424 and 510 of Bdp1 is required for its recruitment by DmSNAPc. Furthermore, to investigate whether the TATA box is required for Bdp1 and TBP recruitment, the U6 TATA box was mutated to an unrelated sequence. Although mutation of the TATA box interfered with the recruitment of TBP by the DmSNAPc‐Bdp1 complex, the TATA mutation did not prevent the recruitment of Bdp1 by DmSNAPc. Interestingly, the non‐conserved amino‐terminal tail of TBP contributed to the efficiency of TBP recruitment by the DmSNAPc‐Bdp1 complex.A body of previous work from our lab has shown that DmSNAPc binds to U6 (Pol III transcribed) and U1 promoters (Pol II transcribed) in distinct conformations. Interestingly, when we switched the U6 proximal sequence element A (PSEA) to a U1 PSEA by a 5‐nucleotide change, DmSNAPc was unable to recruit Bdp1. This finding suggests that a surface of DmSNAPc that interacts with Bdp1 may be occluded when DmSNAPc binds to a U1 PSEA. It further provides a mechanism for the polymerase specificity seen when comparing the snRNA U1 and U6 genes.Support or Funding InformationThis work was supported by the National Science Foundation and by the California Metabolic Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.)

Arrangement of the 4.5 Myb domain repeats of SNAP190 on the U1 gene promoter

The small nuclear RNA activating protein complex (SNAPc) is a transcription factor essential for expression of genes coding for the spliceosomal snRNAs. 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. 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 Myb repeats. Myb repeats are each ~50 amino acid residues in length and were first identified in 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 determine where each of the 4.5 Myb repeats of DmSNAP190 contact the DNA of the U1 PSEA. The protein is arranged with the more C‐terminal Myb repeats contacting the upstream region of the PSEA and the N‐terminal repeats contacting the PSEA closer to the start site. Moreover, the C‐terminal repeats appear to contact the 5′ half of the PSEA in a manner consistent with those observed for Myb‐DNA complexes, whereas the N‐terminal repeats appear to contact the 3′ half of the PSEA (where DmSNAP50 and DmSNAP43 also bind to the PSEA) in a non‐canonical manner. (Supported by NSF and in part by the California Metabolic Research Foundation.)

A TFIIH-Associated Mediator Head Is a Basal Factor of Small Nuclear Spliced Leader RNA Gene Transcription in Early-Diverged Trypanosomes

Genome annotation suggested that early-diverged kinetoplastids possess a reduced set of basal transcription factors. More recent work, however, on the lethal parasite Trypanosoma brucei identified extremely divergent orthologs of TBP, TFIIA, TFIIB, and TFIIH which, together with the small nuclear RNA-activating protein complex, form a transcription preinitiation complex (PIC) at the spliced leader (SL) RNA gene (SLRNA) promoter. The SL RNA is a small nuclear RNA and a trans splicing substrate for the maturation of all pre-mRNAs which is metabolized continuously to sustain gene expression. Here, we identified and biochemically characterized a novel TFIIH-associated protein complex in T. brucei (Med-T) consisting of nine subunits whose amino acid sequences are conserved only among kinetoplastid organisms. Functional analyses in vivo and in vitro demonstrated that the complex is essential for cell viability, SLRNA transcription, and PIC integrity. Molecular structure analysis of purified Med-T and Med-T/TFIIH complexes by electron microscopy revealed that Med-T corresponds to the mediator head module of higher eukaryotes. These data therefore show that mediator is a basal factor for small nuclear SL RNA gene transcription in trypanosomes and that the basal transcription function of mediator head is a characteristic feature of eukaryotes which developed early in their evolution.

Mapping SNAPc subunit domains that interact with specific nucleotide positions in the U1 and U6 snRNA gene promoters

The small nuclear RNA activating protein complex (SNAPc) is essential for transcription of genes coding for the snRNAs (U1, U2, etc.). In D. melanogaster, the heterotrimeric DmSNAPc recognizes a 21 base‐pair DNA sequence, the PSEA, located approximately 40–60 base pairs upstream of the transcription start site. Upon binding the PSEA, DmSNAPc establishes RNA polymerase II pre‐initiation complexes on U1‐U5 promoters but RNA polymerase III pre‐initiation complexes on U6 promoters. Minor differences in nucleotide sequence of the U1 and U6 PSEAs determine RNA polymerase specificity; moreover, DmSNAPc adopts different conformations on these different PSEAs. We have proposed that such conformational differences in DmSNAPc play a key role in determining the different polymerase specificities of the U1 and U6 promoters. To better understand the structure of DmSNAPc‐PSEA complexes, we have developed a novel protocol that combines site‐specific protein‐DNA photo‐cross‐linking with chemical cleavage of the protein. This has allowed us to map regions of the three subunits that contact specific nucleotide positions within the U1 and U6 PSEAs. These data help to establish the orientation of each DmSNAPc subunit on the DNA and have revealed cases in which different domains of the subunits differentially contact the U1 versus U6 PSEAs. (Supported by NSF and in part by the California Metabolic Research Foundation.)

A Map of Drosophila melanogaster Small Nuclear RNA-activating Protein Complex (DmSNAPc) Domains Involved in Subunit Assembly and DNA Binding

Transcription of genes coding for the small nuclear RNAs (snRNAs) is dependent upon a unique transcription factor known as the small nuclear RNA-activating protein complex (SNAPc). SNAPc binds to an essential proximal sequence element located about 40-65 base pairs upstream of the snRNA transcription start site. In the fruit fly Drosophila melanogaster, DmSNAPc contains three distinct polypeptides (DmSNAP190, DmSNAP50, and DmSNAP43) that are stably associated with each other and bind to the DNA as a complex. We have used mutational analysis to identify domains within each subunit that are involved in complex formation with the other two subunits in vivo. We have also identified domains in each subunit required for sequence-specific DNA binding. With one exception, domains required for subunit-subunit interactions lie in the most evolutionarily conserved regions of the proteins. However, DNA binding by DmSNAPc is dependent not only upon the conserved regions but is also highly dependent upon domains outside the conserved regions. Comparison with functional domains identified in human SNAPc indicates many parallels but also reveals significant differences in this ancient yet rapidly evolving system.

Structural and functional studies of the Drosophila melanogaster small nuclear RNA activating protein complex (DmSNAPc)

The small nuclear RNA activating protein complex (SNAPc) is an evolutionarily conserved multi‐subunit factor required for transcription of the spliceosomal small nuclear RNA (snRNA) genes by both RNA polymerase II (U1, U2, U4, and U5) and RNA polymerase III (U6). Three distinct polypeptides have been identified as subunits of D. melanogaster SNAPc; however, the stoichiometry of these three subunits in DmSNAPc had not been investigated. By co‐expressing each subunit with two different tags and by doing band‐shift and super‐shift analyses, we have determined that DmSNAPc is a heterotrimer with a 1:1:1 subunit stoichiometry. DmSNAPc recognizes an ~21 bp long DNA sequence denoted the PSEA about 40‐60 bp upstream of the transcription start site. Interestingly, the PSEAs of the U1 and U6 genes are not interchangeable even though they are identical at 16 of 21 nucleotide positions. In fact, changing the U1 PSEA to a U6 PSEA inactivated the U1 promoter. We have now found that this substitution does not affect the association of DmSNAPc with the promoter; instead, it disrupts the recruitment of TBP. This finding is consistent with a working model in which DmSNAPc binds in different conformations to the U1 and U6 PSEAs. We believe these conformational differences in DmSNAPc lead to differential RNA polymerase selectivity at the U1 and U6 promoters. Supported by NSF and in part by the California Metabolic Research Foundation.

Subunit stoichiometry of the Drosophila melanogaster small nuclear RNA activating protein complex (SNAPc)

Small nuclear RNA activating protein complex (SNAPc) is a multi-subunit transcription factor required for expression of small nuclear RNA genes. This protein binds to a promoter element located approximately 40–65bp upstream of the transcription start site. In Drosophila melanogaster, DmSNAPc contains three distinct polypeptide subunits: DmSNAP190, DmSNAP50, and DmSNAP43. The subunit stoichiometry in SNAPc complexed with DNA has not been examined. Therefore, the ability of differently tagged but otherwise identical subunits to associate with each other into the same protein–DNA complex was assayed by antibody super-shift analysis. The results reveal that DmSNAPc contains only a single copy of each of the three subunits. Structured summaryMINT-6788068:DmSNAP43 (uniprotkb:Q9VF25), DmSNAP50 (uniprotkb:Q7JUY8) and DmSNAP190 (uniprotkb:Q9VHX0) physically interact (MI:0218) by electrophoretic mobility shift assay (MI:0413)MINT-6788151:DmSNAP190 (uniprotkb:Q9VHX0), DmSNAP50 (uniprotkb:Q7JUY8) and DmSNAP43 (uniprotkb:Q9VF25) physically interact (MI:0218) by molecular sieving (MI:0071)

Mitotic Functions for SNAP45, a Subunit of the Small Nuclear RNA-activating Protein Complex SNAPc

The small nuclear RNA-activating protein complex SNAPc is required for transcription of small nuclear RNA genes and binds to a proximal sequence element in their promoters. SNAPc contains five types of subunits stably associated with each other. Here we show that one of these polypeptides, SNAP45, also known as PTF δ, localizes to centrosomes during parts of mitosis, as well as to the spindle midzone during anaphase and the mid-body during telophase. Consistent with localization to these mitotic structures, both down- and up-regulation of SNAP45 lead to a G2/M arrest with cells displaying abnormal mitotic structures. In contrast, down-regulation of SNAP190, another SNAPc subunit, leads to an accumulation of cells with a G0/G1 DNA content. These results are consistent with the proposal that SNAP45 plays two roles in the cell, one as a subunit of the transcription factor SNAPc and another as a factor required for proper mitotic progression.

DmSNAPc domains for subunit assembly and DNA binding

The small nuclear RNA activating protein complex (SNAPc) is the major unique transcription factor required for transcription of genes coding for small nuclear RNAs (snRNAs). In the fruit fly Drosophila melanogaster, DmSNAPc contains three distinct subunits (DmSNAP190, DmSNAP50, and DmSNAP43) that form a complex before binding to an snRNA gene promoter. We have used mutational analysis to identify domains within each subunit of DmSNAPc that are required for complex formation with the other two subunits in vivo. Also, we mapped domains in each subunit that are required for the DNA‐binding activity of DmSNAPc. We have found that the most evolutionarily conserved regions of the proteins are involved in SNAP complex assembly. Nevertheless, we found that domains outside of the conserved regions are also important for the DNA binding activity of DmSNAPc, even though they are not required for subunit assembly. Comparing our findings with published results in the human system indicates not only many important similarities but also significant differences in this ancient though rapidly evolving system. This work is supported by National Science Foundation grants MCB‐0131151 and MCB‐0641350 and in part by the California Metabolic Research Foundation. M. T. is a recipient of an Arne N. Wick Pre‐doctoral Research Fellowship from the California Metabolic Research Foundation.

Spliced Leader RNA Gene Transcription in Trypanosoma brucei Requires Transcription Factor TFIIH

Trypanosomatid parasites share a gene expression mode which differs greatly from that of their human and insect hosts. In these unicellular eukaryotes, protein-coding genes are transcribed polycistronically and individual mRNAs are processed from precursors by spliced leader (SL) trans splicing and polyadenylation. In trans splicing, the SL RNA is consumed through a transfer of its 5'-terminal part to the 5' end of mRNAs. Since all mRNAs are trans spliced, the parasites depend on strong and continuous SL RNA synthesis mediated by RNA polymerase II. As essential factors for SL RNA gene transcription in Trypanosoma brucei, the general transcription factor (GTF) IIB and a complex, consisting of the TATA-binding protein-related protein 4, the small nuclear RNA-activating protein complex, and TFIIA, were recently identified. Although T. brucei TFIIA and TFIIB are extremely divergent to their counterparts in other eukaryotes, their characterization suggested that trypanosomatids do form a class II transcription preinitiation complex at the SL RNA gene promoter and harbor orthologues of other known GTFs. TFIIH is a GTF which functions in transcription initiation, DNA repair, and cell cycle control. Here, we investigated whether a T. brucei TFIIH is important for SL RNA gene transcription and found that silencing the expression of the highly conserved TFIIH subunit XPD in T. brucei affected SL RNA gene synthesis in vivo, and depletion of this protein from extract abolished SL RNA gene transcription in vitro. Since we also identified orthologues of the TFIIH subunits XPB, p52/TFB2, and p44/SSL1 copurifying with TbXPD, we concluded that the parasite harbors a TFIIH which is indispensable for SL RNA gene transcription.

Highly Efficient Tandem Affinity Purification of Trypanosome Protein Complexes Based on a Novel Epitope Combination

Tandem affinity purification (TAP) allows for rapid and efficient purification of epitope-tagged protein complexes from crude extracts under native conditions. The method was established in yeast and has been successfully applied to other organisms, including mammals and trypanosomes. However, we found that the original method, which is based on the TAP tag, consisting of a duplicate protein A epitope, a tobacco etch virus protease cleavage site, and the calmodulin-binding peptide (CBP), did not yield enough recovery of transcription factor SNAPc (for small nuclear RNA-activating protein complex) from crude trypanosome extracts for protein identification. Specifically, the calmodulin affinity chromatography step proved to be inefficient. To overcome this problem, we replaced CBP by the protein C epitope (ProtC) and termed this new epitope combination PTP tag. ProtC binds with high affinity to the monoclonal antibody HPC4, which has the unique property of requiring calcium for antigen recognition. Thus, analogous to the calcium-dependent CBP-calmodulin interaction, ProtC-tagged proteins can be released from immobilized HPC4 by a chelator of divalent cations. While this property was retained, epitope substitution improved purification in our experiments by eliminating the inefficiency of calmodulin affinity chromatography and by providing an alternative way of elution using the ProtC peptide in cases where EGTA inactivated protein function. Furthermore, HPC4 allowed highly sensitive and specific detection of ProtC-tagged proteins after protease cleavage. Thus far, we have successfully purified and characterized the U1 small nuclear ribonucleoprotein particle, the transcription factor complex TATA-binding protein related factor 4 (TRF4)/SNAPc/transcription factor IIA (TFIIA), and RNA polymerase I of Trypanosoma brucei.

Characterization of a multisubunit transcription factor complex essential for spliced-leader RNA gene transcription in Trypanosoma brucei.

In the unicellular human parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., the spliced-leader (SL) RNA is a key molecule in gene expression donating its 5'-terminal region in SL addition trans splicing of nuclear pre-mRNA. While there is no evidence that this process exists in mammals, it is obligatory in mRNA maturation of trypanosomatid parasites. Hence, throughout their life cycle, these organisms crucially depend on high levels of SL RNA synthesis. As putative SL RNA gene transcription factors, a partially characterized small nuclear RNA-activating protein complex (SNAP(c)) and the TATA-binding protein related factor 4 (TRF4) have been identified thus far. Here, by tagging TRF4 with a novel epitope combination termed PTP, we tandem affinity purified from crude T. brucei extracts a stable and transcriptionally active complex of six proteins. Besides TRF4 these were identified as extremely divergent subunits of SNAP(c) and of transcription factor IIA (TFIIA). The latter finding was unexpected since genome databases of trypanosomatid parasites appeared to lack general class II transcription factors. As we demonstrate, the TRF4/SNAP(c)/TFIIA complex binds specifically to the SL RNA gene promoter upstream sequence element and is absolutely essential for SL RNA gene transcription in vitro.

Cooperation between Small Nuclear RNA-activating Protein Complex (SNAPC) and TATA-box-binding Protein Antagonizes Protein Kinase CK2 Inhibition of DNA Binding by SNAPC

Protein kinase CK2 regulates RNA polymerase III transcription of human U6 small nuclear RNA (snRNA) genes both negatively and positively depending upon whether the general transcription machinery or RNA polymerase III is preferentially phosphorylated. Human U1 snRNA genes share similar promoter architectures as that of U6 genes but are transcribed by RNA polymerase II. Herein, we report that CK2 inhibits U1 snRNA gene transcription by RNA polymerase II. Decreased levels of endogenous CK2 correlates with increased U1 expression, whereas CK2 associates with U1 gene promoters, indicating that it plays a direct role in U1 gene regulation. CK2 phosphorylates the general transcription factor small nuclear RNA-activating protein complex (SNAP(C)) that is required for both RNA polymerase II and III transcription, and SNAP(C) phosphorylation inhibits binding to snRNA gene promoters. However, restricted promoter access by phosphorylated SNAP(C) can be overcome by cooperative interactions with TATA-box-binding protein at a U6 promoter but not at a U1 promoter. Thus, CK2 may have the capacity to differentially regulate U1 and U6 transcription even though SNAP(C) is universally utilized for human snRNA gene transcription.

RNA polymerase II-dependent transcription in trypanosomes is associated with a SNAP complex-like transcription factor.

Spliced leader RNA transcription is essential for cell viability in trypanosomes. The SL RNA genes are expressed from the only defined RNA polymerase II-dependent promoter identified to date in the trypanosome genome. The SL RNA gene promoter has been shown by in vitro and in vivo analyses to have a tripartite architecture. The upstream most cis-acting element, called PBP-1E, is located between 70 and 60 bp upstream from the transcription start site. This essential element functions along with two downstream elements to direct efficient and proper initiation of transcription. Electrophoretic mobility-shift studies detected a 122-kDa protein, called PBP-1, which interacts with PBP-1E. This protein is the first sequence-specific, double-stranded DNA-binding protein isolated in trypanosomes. Three polypeptides copurify with PBP-1 activity, suggesting that PBP-1 is composed of 57-, 46-, and 36-kDa subunits. We have cloned the genes that encode the 57- and 46-kDa subunits. The 46-kDa protein is a previously uncharacterized protein and may be unique to trypanosomes. Its predicted tertiary structure suggests it binds DNA as part of a complex. The 57-kDa subunit is orthologous to the human small nuclear RNA-activating protein (SNAP)50, which is an essential subunit of the SNAP complex (SNAPc). In human cells, SNAPc binds to the proximal sequence element in both RNA polymerase II- and III-dependent small nuclear RNA gene promoters. These findings identify a surprising link in the transcriptional machinery across a large evolutionary distance in the regulation of small nuclear RNA genes in eukaryotes.