U6 snRNA Gene Research Articles

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.

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.)

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.

Differentially expressed, variant U1 snRNAs regulate gene expression in human cells

Human U1 small nuclear (sn)RNA, required for splicing of pre-mRNA, is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes, also located on chromosome 1 (1q12-21), were thought to be pseudogenes. However, many of these “variant” (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells, we have identified global transcriptome changes following interrogation of the Affymetrix Human Exon ST 1.0 array. Our results indicate that this vU1 snRNA regulates expression of a subset of target genes at the level of pre-mRNA processing. This is the first indication that variant U1 snRNAs have a biological function in vivo. Furthermore, some vU1 snRNAs are packaged into unique ribonucleoproteins (RNPs), and many vU1 snRNA genes are differentially expressed in human embryonic stem cells (hESCs) and HeLa cells, suggesting developmental control of RNA processing through expression of different sets of vU1 snRNPs.

Evidence for 5S rDNA Horizontal Transfer in the toadfish Halobatrachus didactylus (Schneider, 1801) based on the analysis of three multigene families

BackgroundThe Batrachoididae family is a group of marine teleosts that includes several species with more complicated physiological characteristics, such as their excretory, reproductive, cardiovascular and respiratory systems. Previous studies of the 5S rDNA gene family carried out in four species from the Western Atlantic showed two types of this gene in two species but only one in the other two, under processes of concerted evolution and birth-and-death evolution with purifying selection. Here we present results of the 5S rDNA and another two gene families in Halobatrachus didactylus, an Eastern Atlantic species, and draw evolutionary inferences regarding the gene families. In addition we have also mapped the genes on the chromosomes by two-colour fluorescence in situ hybridization (FISH).ResultsTwo types of 5S rDNA were observed, named type α and type β. Molecular analysis of the 5S rDNA indicates that H. didactylus does not share the non-transcribed spacer (NTS) sequences with four other species of the family; therefore, it must have evolved in isolation. Amplification with the type β specific primers amplified a specific band in 9 specimens of H. didactylus and two of Sparus aurata. Both types showed regulatory regions and a secondary structure which mark them as functional genes. However, the U2 snRNA gene and the ITS-1 sequence showed one electrophoretic band and with one type of sequence. The U2 snRNA sequence was the most variable of the three multigene families studied. Results from two-colour FISH showed no co-localization of the gene coding from three multigene families and provided the first map of the chromosomes of the species.ConclusionsA highly significant finding was observed in the analysis of the 5S rDNA, since two such distant species as H. didactylus and Sparus aurata share a 5S rDNA type. This 5S rDNA type has been detected in other species belonging to the Batrachoidiformes and Perciformes orders, but not in the Pleuronectiformes and Clupeiformes orders. Two hypotheses have been outlined: one is the possible vertical permanence of the shared type in some fish lineages, and the other is the possibility of a horizontal transference event between ancient species of the Perciformes and Batrachoidiformes orders. This finding opens a new perspective in fish evolution and in the knowledge of the dynamism of the 5S rDNA. Cytogenetic analysis allowed some evolutionary trends to be roughed out, such as the progressive change in the U2 snDNA and the organization of (GATA)n repeats, from dispersed to localized in one locus. The accumulation of (GATA)n repeats in one chromosome pair could be implicated in the evolution of a pair of proto-sex chromosomes. This possibility could situate H. didactylus as the most highly evolved of the Batrachoididae family in terms of sex chromosome biology.

Architectural arrangement of the small nuclear RNA (snRNA)-activating protein complex 190 subunit (SNAP190) on U1 snRNA gene promoter DNA.

Myb repeats ∼52 amino acid residues in length were first characterized in the oncogenic Myb transcription factor, which contains three tandem Myb repeats in its DNA-binding domain. Proteins of this family normally contain either one, two, or three tandem Myb repeats that are involved in protein-DNA interactions. The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is a heterotrimeric transcription factor that is required for expression of small nuclear RNA genes. This complex binds to an essential promoter element, the proximal sequence element, centered ∼50 base pairs upstream of the transcription start site of snRNA genes. SNAP190, the largest subunit of SNAPc, uncharacteristically contains 4.5 tandem Myb repeats. Little is known about the arrangement of the Myb repeats in the SNAPc-DNA complex, and it has not been clear whether all 4.5 Myb repeats contact the DNA. By using a site-specific protein-DNA photo-cross-linking assay, we have now mapped specific nucleotides where each of the Myb repeats of Drosophila melanogaster SNAP190 interacts with a U1 snRNA gene proximal sequence element. The results reveal the topological arrangement of the 4.5 SNAP190 Myb repeats relative to the DNA and to each other when SNAP190 is bound to a U1 promoter as a subunit of SNAPc.

C16orf57, a gene mutated in poikiloderma with neutropenia, encodes a putative phosphodiesterase responsible for the U6 snRNA 3′ end modification

C16orf57 encodes a human protein of unknown function, and mutations in the gene occur in poikiloderma with neutropenia (PN), which is a rare, autosomal recessive disease. Interestingly, mutations in C16orf57 were also observed among patients diagnosed with Rothmund-Thomson syndrome (RTS) and dyskeratosis congenita (DC), which are caused by mutations in genes involved in DNA repair and telomere maintenance. A genetic screen in Saccharomyces cerevisiae revealed that the yeast ortholog of C16orf57, USB1 (YLR132C), is essential for U6 small nuclear RNA (snRNA) biogenesis and cell viability. Usb1 depletion destabilized U6 snRNA, leading to splicing defects and cell growth defects, which was suppressed by the presence of multiple copies of the U6 snRNA gene SNR6. Moreover, Usb1 is essential for the generation of a unique feature of U6 snRNA; namely, the 3'-terminal phosphate. RNAi experiments in human cells followed by biochemical and functional analyses confirmed that, similar to yeast, C16orf57 encodes a protein involved in the 2',3'-cyclic phosphate formation at the 3' end of U6 snRNA. Advanced bioinformatics predicted that C16orf57 encodes a phosphodiesterase whose putative catalytic activity is essential for its function in vivo. Our results predict an unexpected molecular basis for PN, DC, and RTS and provide insight into U6 snRNA 3' end formation.

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.

First approach to studying the genetics of the meagre (Argyrosomus regius; Asso, 1801) using three multigene families

The meagre (Argyrosomus regius) is a commercially important species for fisheries, and aquaculture production has been increasing in recent years. However, this growing importance has not been matched by increased genetic knowledge of the species. For this reason, an initial approach has been made to understanding the genetics of the meagre, using three particular multigene families (45S and 5S ribosomal DNA and U2 snRNA gene) as both molecular and cytogenetic markers. Only one type of 5S rDNA and only one ITS-1 (from 45S rDNA) were obtained, and these therefore could provide the basis for the development of genetic markers for this species. However, the U2 snRNA gene produced a multi-band electrophoretic pattern, and some of these bands were demonstrated to be linked with the U1 snRNA gene. This linkage could also provide a good species-specific marker which could be useful for tracing the evolutionary history of the Sciaenidae family. In this study, we have described for the first time the karyotype of the meagre: this is composed of 48 acrocentric chromosomes. Each of the three gene families were localized on different chromosome pairs, although the U2 snRNA gene probe was also located scattered throughout the genome of the meagre, as expected by the amplification pattern.

Genomic organization and comparative chromosome mapping of the U1 snRNA gene in cichlid fish, with an emphasis in Oreochromis niloticus

To better understand genomic and chromosomal organization and evolutionary patterns of the U1 snRNA gene in cichlid fish, the gene was cytogenetically mapped and comparatively analyzed in 19 species belonging to several clades of the group. Moreover, the distribution and organization of U1 snRNA gene was analyzed in the Oreochromis niloticus genome. The results indicated high conservation of one chromosomal cluster of U1 snRNA in the African, Asian, and South American species, with few variations in the chromosomal position of the clusters in the South American species. The genomic analysis of U1 revealed a distinct scenario from that observed under the cytogenetic mapping. An enrichment of the U1 gene on linkage group (LG) 14 was observed that did not correspond to the same chromosome that harbors the U1 cluster identified by cytogenetic mapping. Moreover, it was revealed that the presence of several distinct transposable elements in the U1 gene flanking regions could be involved in the spreading of this sequence, but the generation of new, large snRNA clusters (detectable by fluorescent in situ hybridization, FISH) is apparently hampered. These results contribute to the understanding of multigene families' evolution and reinforce the utility of integrative analysis and the use of cytogenetic and bioinformatic methods to address the genomic and chromosomal evolutionary patterns of repeated DNAs among vertebrates. Moreover, the U1 gene represents a useful new chromosomal marker for cytogenetic studies.

Mutation of a U2 snRNA Gene Causes Global Disruption of Alternative Splicing and Neurodegeneration

Although uridine-rich small nuclear RNAs (U-snRNAs) are essential for pre-mRNA splicing, little is known regarding their function in the regulation of alternative splicing or of the biological consequences of their dysfunction in mammals. Here, we demonstrate that mutation of Rnu2-8, one of the mouse multicopy U2 snRNA genes, causes ataxia and neurodegeneration. Coincident with the observed pathology, the level of mutant U2 RNAs was highest in the cerebellum and increased after granule neuron maturation. Furthermore, neuron loss was strongly dependent on the dosage of mutant and wild-type snRNA genes. Comprehensive transcriptome analysis identified a group of alternative splicing events, including the splicing of small introns, which were disrupted in the mutant cerebellum. Our results suggest that the expression of mammalian U2 snRNA genes, previously presumed to be ubiquitous, is spatially and temporally regulated, and dysfunction of a single U2 snRNA causes neuron degeneration through distortion of pre-mRNA splicing.

Different domains of SNAPc subunits closely interact with U1 versus U6 snRNA gene promoter sequences for differential recruitment of RNA polymerases II and III

Different domains of SNAPc subunits closely interact with U1 versus U6 snRNA gene promoter sequences for differential recruitment of RNA polymerases II and III

Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and U2 snRNA gene by double-colour FISH in species of the Batrachoididae family

In the present study dual-colour fluorescence in situ hybridization (FISH) was performed to study the chromosomal distribution of 18S and 5S rDNAs, (GATA)(n) and 5S rDNA, and U2 snRNA and 18S rDNA in four species of Batrachoididae family: Amphichthys cryptocentrus, Batrachoides manglae, Porichthys plectrodon and Thalassophryne maculosa. The 18S rDNA signals were present in only one pair of chromosomes in all the four Batrachoididae species. The 5S rDNA was mapped on one pair of chromosomes, except in B. manglae, which showed a hybridization signal in two pairs. The two ribosomal genes are located on different chromosome pairs, except in A. cryptocentrus, in which they appear co-located. In all the cases, the (GATA)(n) probe produced disperse hybridization signals in all four species. The U2 snRNA signals appear very widely scattered in A. cryptocentrus, P. plectrodon, but show a degree of clustering in a specific chromosome pair in B. manglae. In T. maculosa, they are thinly dispersed and strong hybridization signals are observed co-located to the 18S rDNA-bearing chromosomes. Finally, a double-colour FISH with U2 snRNA and 5S rDNA probes was performed in B. manglae, and this showed that these genes were not co-located. These results have been compared with those from another Batrachoididae species, and evolutive processes of these species are discussed.

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.)

Inhibition of U6 snRNA Transcription by PTEN.

PROBLEM STATEMENT: RNA polymerase III (RNA pol III) is responsible for transcribing many of the small structural RNA molecules involved in RNA processing and protein translation, thereby regulating the growth rate of a cell. RNA pol III transcribes both gene internal (tRNA) and gene external (U6 snRNA) promoters and proper initiation by RNA polymerase III requires the transcription initiation factor TFIIIB. TFIIIB has been shown to be a target of repression by tumor suppressors such as ARF, p53, RB and the RB-related pocket proteins. Also, TFIIIB activity is stimulated by the oncogenes c-Myc and the ERK mitogen-activated protein kinase. Recently, two TFIIIB subunits, BRF1 and BRF2, have been demonstrated to behave as oncogenes, making deregulation of TFIIIB activity and thus RNA pol III transcription an important step in tumor development. PTEN is a commonly mutated tumor suppressor regulating cell growth, proliferation and survival. Thus, we sought to examine the potential role of PTEN in regulating U6 snRNA transcription. APPROACH: We examined the potential for PTEN to regulate U6 snRNA transcription using in vitro RNA pol III luciferase assays, western blotting and deletion analysis in cancer cell lines differing in their PTEN status. RESULTS: Using breast, cervical, prostate and glioblastoma cancer cells we demonstrate: (1) PTEN inhibition of gene external RNA pol III transcription is cell type specific, (2) PTEN-mediated inhibition of U6 transcription occurs via the C2 lipid-binding domain and (3) PTEN repression of U6 transcription occurs, at least in part, through the TFIIIB subunit BRF2. CONCLUSION/RECOMMENDATIONS: Our data demonstrates that regulation of the U6 snRNA gene by PTEN is mediated, in part by the TFIIIB oncogene BRF2, potentially identifying novel targets for chemotherapeutic drug design.

Cleavage-induced termination in U2 snRNA gene expression

The maturation of many small nuclear RNAs is dependent on RNase III-like endonuclease mediated cleavage, which generates a loading site for the exosome complex that trims the precursor at its 3′ end. Using a temperature sensitive Pac1 nuclease, here we show that the endonuclease cleavage is equally important in terminating the transcription of the U2 snRNA in Schizosaccharomyces pombe. Using a temperature sensitive Dhp1p 5′ → 3′ exonuclease, we demonstrate that it also is an essential component of the termination pathway. Taken together the results support a “reversed torpedoes” model for the termination and maturation of the U2 snRNA; the Pac1 endonuclease cleavage provides entry sites for the 3′ and 5′ exonuclease activities, leading to RNA maturation in one direction and transcript termination in the other.

Chromatin structure is implicated in "late" elongation checkpoints on the U2 snRNA and beta-actin genes.

The negative elongation factor NELF is a key component of an early elongation checkpoint generally located within 100 bp of the transcription start site of protein-coding genes. Negotiation of this checkpoint and conversion to productive elongation require phosphorylation of the carboxy-terminal domain of RNA polymerase II (pol II), NELF, and DRB sensitivity-inducing factor (DSIF) by positive transcription elongation factor b (P-TEFb). P-TEFb is dispensable for transcription of the noncoding U2 snRNA genes, suggesting that a NELF-dependent checkpoint is absent. However, we find that NELF at the end of the 800-bp U2 gene transcription unit and RNA interference-mediated knockdown of NELF causes a termination defect. NELF is also associated 800 bp downstream of the transcription start site of the beta-actin gene, where a "late" P-TEFb-dependent checkpoint occurs. Interestingly, both genes have an extended nucleosome-depleted region up to the NELF-dependent control point. In both cases, transcription through this region is P-TEFb independent, implicating chromatin in the formation of the terminator/checkpoint. Furthermore, CTCF colocalizes with NELF on the U2 and beta-actin genes, raising the possibility that it helps the positioning and/or function of the NELF-dependent control point on these genes.

A Genetic Screen for Suppressors of a Mutated 5′ Splice Site Identifies Factors Associated With Later Steps of Spliceosome Assembly

Many alleles of human disease genes have mutations within splicing consensus sequences that activate cryptic splice sites. In Caenorhabditis elegans, the unc-73(e936) allele has a G-to-U mutation at the first base of the intron downstream of exon 15, which results in an uncoordinated phenotype. This mutation triggers cryptic splicing at the -1 and +23 positions and retains some residual splicing at the mutated wild-type (wt) position. We previously demonstrated that a mutation in sup-39, a U1 snRNA gene, suppresses e936 by increasing splicing at the wt splice site. We report here the results of a suppressor screen in which we identify three proteins that function in cryptic splice site choice. Loss-of-function mutations in the nonessential splicing factor smu-2 suppress e936 uncoordination through changes in splicing. SMU-2 binds SMU-1, and smu-1(RNAi) also leads to suppression of e936. A dominant mutation in the conserved C-terminal domain of the C. elegans homolog of the human tri-snRNP 27K protein, which we have named SNRP-27, suppresses e936 uncoordination through changes in splicing. We propose that SMU-2, SMU-1, and SNRP-27 contribute to the fidelity of splice site choice after the initial identification of 5' splice sites by U1 snRNP.

O-GlcNAc modification of Sp1 inhibits the functional interaction between Sp1 and Oct1

Sp1 is a ubiquitous transcription factor that is modified by multiple O-linked N-acetylglucosamines (O-GlcNAc). Previously, O-GlcNAcylation of a specific site of Sp1 was shown to inhibit Sp1 transcriptional activity. Yet, how O-GlcNAc on other modification sites affects Sp1 function and how O-GlcNAcylation of Sp1 affects the transcriptional regulation of a target gene remains unknown. Here we show that O-GlcNAc within the second serine/threonine-rich region of Sp1 interrupts a known interaction between Sp1 and Oct1, and inhibits the cooperative activation of the U2 snRNA gene by Sp1 and Oct1. Structured summary MINT- 6803452: Sp1 (uniprotkb- P08047) physically interacts (MI: 0218) with Oct1 (uniprotkb: P14859) by anti tag coimmunoprecipitation (MI: 0007) MINT- 6803426, MINT- 6803438: Oct1 (uniprotkb:P14859) binds (MI: 0407) to Sp1 (uniprotkb: P08047) by pull down (MI: 0096) MINT- 6803470, MINT- 6803484: Sp1 (uniprotkb: P08047) physically interacts (MI: 0218) with Oct1 (uniprotkb: P14859) by anti bait coimmunoprecipitation (MI: 0006)