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![Pol II CTD phosphorylation events in snRNA gene transcription. Initially, the cyclin-dependent kinase (CDK)7 subunit of TFIIH phosphorylates Ser5 and Ser7. RPAP2 interacts with Ser7P. RPAP2 in turn recruits the Integrator subunits Int1, Int4, Int5, Int6 and Int7. RPAP2 dephosphorylates Ser5P, and positive-transcription elongation factor b (P-TEFb) is recruited by a mechanism still unknown. The P-TEFb subunit CDK9 phosphorylates Ser2. The double phosphorylation on Ser2 and Ser7 recruits Int9/11, which allows RNA processing to occur [62].](publication/317584554/figure/fig3/AS:1165367078588417@1654856571819/Pol-II-CTD-phosphorylation-events-in-snRNA-gene-transcription-Initially-the.jpeg)
Pol II CTD phosphorylation events in snRNA gene transcription. Initially, the cyclin-dependent kinase (CDK)7 subunit of TFIIH phosphorylates Ser5 and Ser7. RPAP2 interacts with Ser7P. RPAP2 in turn recruits the Integrator subunits Int1, Int4, Int5, Int6 and Int7. RPAP2 dephosphorylates Ser5P, and positive-transcription elongation factor b (P-TEFb) is recruited by a mechanism still unknown. The P-TEFb subunit CDK9 phosphorylates Ser2. The double phosphorylation on Ser2 and Ser7 recruits Int9/11, which allows RNA processing to occur [62].
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In addition to protein-coding genes, RNA polymerase II (pol II) transcribes numerous genes for non-coding RNAs, including the small-nuclear (sn)RNA genes. snRNAs are an important class of non-coding RNAs, several of which are involved in pre-mRNA splicing. The molecular mechanisms underlying expression of human pol II-transcribed snRNA genes are le...
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Despite significant steps in our understanding of Alzheimer's disease (AD), many of the molecular processes underlying its pathogenesis remain largely unknown. Here, we focus on the role of non-coding RNAs produced by small interspersed nuclear elements (SINEs). RNAs from SINE B2 repeats in mouse and SINE Alu repeats in humans, long regarded as "ju...
Citations
... The other gene belonging to the GS-2 NCBP3 is much less studied in cancer. It's a non-canonical cap bonding protein directly implicated in various steps of gene expression, likely to regulate pre-mRNA splicing, pre-mRNA 3'-end processing, mRNA nuclear export, and mRNA translation (Baltz et al. 2012;Gebhardt et al. 2015;Guiro and Murphy 2017;Li et al. 2017;Dou et al. 2020). Current literature indicates that NCBP3 is a non-essential protein under basal conditions and appears functionally critical under pathological conditions that include viral infections and cancer (Gebhardt et al. 2015(Gebhardt et al. , 2019Zhang et al. 2019). ...
Medulloblastoma (MB) is one of the most common pediatric brain tumors and it is estimated that one-third of patients will not achieve long-term survival. Conventional prognostic parameters have limited and unreliable correlations with MB outcome, presenting a major challenge for patients’ clinical improvement. Acknowledging this issue, our aim was to build a gene signature and evaluate its potential as a new prognostic model for patients with the disease. In this study, we used six datasets totaling 1679 samples including RNA gene expression and DNA methylation data from primary MB as well as control samples from healthy cerebellum. We identified methylation-driven genes (MDGs) in MB, genes whose expression is correlated with their methylation. We employed LASSO regression, incorporating the MDGs as a parameter to develop the prognostic model. Through this approach, we derived a two-gene signature (GS-2) of candidate prognostic biomarkers for MB (CEMIP and NCBP3). Using a risk score model, we confirmed the GS-2 impact on overall survival (OS) with Kaplan-Meier analysis. We evaluated its robustness and accuracy with receiver operating characteristic curves predicting OS at 1, 3, and 5 years in multiple independent datasets. The GS-2 showed highly significant results as an independent prognostic biomarker compared to traditional MB markers. The methylation-regulated GS-2 risk score model can effectively classify patients with MB into high and low-risk, reinforcing the importance of this epigenetic modification in the disease. Such genes stand out as promising prognostic biomarkers with potential application for MB treatment.
... Processing of the nascent transcript at the 3´-end requires the 3´-box that is located downstream of the coding sequence (Hernandez, 1985). These regulatory elements differ considerably in the vU1 genes and could be one of the underlying reasons for their low expression levels (Guiro & Murphy, 2017;Guiro & O'Reilly, 2015). Additionally, the vU1s may not be appropriately recognized by nuclear 3´-end processing machinery and instead be targeted for degradation (Lardelli & Lykke-Andersen, 2020). ...
The human U1 snRNA is encoded by a multigene family consisting of transcribed variants and defective pseudogenes. Many variant U1 (vU1) snRNAs have been demonstrated to not only be transcribed but also processed by the addition of a trimethylated guanosine cap, packaged into snRNPs, and assembled into spliceosomes, however, their capacity to facilitate pre-mRNA splicing has, so far, not been tested. A recent systematic analysis of the human snRNA genes identified 178 U1 snRNA genes that are present in the genome as either tandem arrays or single genes on multiple chromosomes. Of these, 15 were found to be expressed in human tissues and cell lines, although at significantly low levels from their endogenous loci, less than 0.001% of the canonical U1 snRNA. In this study, we found that placing the variants in the context of the regulatory elements of the RNU1-1 gene improves expression of many variants to levels comparable to the canonical U1 snRNA. Application of a previously established HeLa cell based minigene reporter assay to examine the capacity of the vU1 snRNAs to support pre-mRNA splicing revealed that even though the exogenously expressed variant snRNAs were enriched in the nucleus, only a few had a measurable effect on splicing.
... However, there was an interesting observation while analysing the transcriptomic data from the pituitary samples. Small nuclear RNAs (snRNAs) such as U1, U2 and U4, that form a key component of the spliceosomal machinery in the eukaryotes [31,32], were found to be differentially expressed in pituitary, between the two euthanasia methods. These snRNAs were found to be significantly higher in pituitary samples belonging to the ANOXIA group in comparison to the T61 group. ...
Background
Transcriptomic studies often require collection of fresh tissues post euthanasia. The chosen euthanasia method might have the potential to induce variations in gene expressions that are unlinked with the experimental design. The present study compared the suitability of ‘nitrogen gas in foam’ (ANOXIA) in comparison to a non-barbiturate anaesthetic, T-61® (T61), for euthanizing piglets used in transcriptome research. Further, the effect of common tissue storage conditions, RNAlater™ (RL) and snap freezing in liquid nitrogen (LN2), on gene expression profiles were also analysed.
Results
On comparison of the 3’mRNA-Seq data generated from pituitary, hypothalamus, liver and lung tissues, no significant differential expression in the protein coding genes were detected between the euthanasia methods. This implies that the nitrogen anoxia method could be a suitable alternative for euthanasia of piglets used in transcriptomic research. However, small nuclear RNAs (snRNAs) that constitute the eukaryotic spliceosomal machinery were found to be significantly higher (log2fold change ≥ 2.0, and adjusted p value ≤ 0.1) in pituitary samples collected using ANOXIA. Non-protein coding genes like snRNAs that play an important role in pre-mRNA splicing can subsequently modify gene expression. Storage in RL was found to be superior in preserving RNA compared to LN2 storage, as evidenced by the significantly higher RIN values in representative samples. However, storage in RL as opposed to LN2, also influenced differential gene expression in multiple tissues, perhaps as a result of its inability to inhibit biological activity during storage. Hence such external sources of variations should be carefully considered before arriving at research conclusions.
Conclusions
Source of biological variations like euthanasia method and storage condition can confound research findings. Even if we are unable to prevent the effect of these external factors, it will be useful to identify the impact of these variables on the parameter under observation and thereby prevent misinterpretation of our results.
... First, we found that the mutant cells contain 3 -extended U4atac108 126del and U4atac111G>A transcripts. Accordingly, misprocessed and uncleaved forms of snRNAs as well as accumulation of aberrant polyadenylated U1 transcripts is a hallmark of a defective Integrator function in 3 -end processing of snRNAs (58)(59)(60). Second, it has also been shown that terminal stem-loops within U2 and U7 snRNAs promote 3 -end processing (61,62). ...
Various genetic diseases associated with microcephaly and developmental defects are due to pathogenic variants in the U4atac small nuclear RNA (snRNA), a component of the minor spliceosome essential for the removal of U12-type introns from eukaryotic mRNAs. While it has been shown that a few RNU4ATAC mutations result in impaired binding of essential protein components, the molecular defects of the vast majority of variants are still unknown. Here, we used lymphoblastoid cells derived from RNU4ATAC compound heterozygous (g.108_126del;g.111G>A) twin patients with MOPD1 phenotypes to analyze the molecular consequences of the mutations on small nuclear ribonucleoproteins (snRNPs) formation and on splicing. We found that the U4atac108_126del mutant is unstable and that the U4atac111G>A mutant as well as the minor di- and tri-snRNPs are present at reduced levels. Our results also reveal the existence of 3'-extended snRNA transcripts in patients' cells. Moreover, we show that the mutant cells have alterations in splicing of INTS7 and INTS10 minor introns, contain lower levels of the INTS7 and INTS10 proteins and display changes in the assembly of Integrator subunits. Altogether, our results show that compound heterozygous g.108_126del;g.111G>A mutations induce splicing defects and affect the homeostasis and function of the Integrator complex.
... In contrast to the Pol III-dependent snRNA promoter, Pol II-dependent snRNA promoters lack a TATA box motif 7 . To produce snRNAs, Pol II uses many of its accessory factors that are used for mRNA synthesis, but it additionally requires specific factors for transcription initiation and elongation 8 . ...
... The intervening genomic region between the PSE and Article https://doi.org/10.1038/s41594-022-00857-w DSE may be decorated by a nucleosome 8 . In the future, our work may be expanded to study how DSE binding activators interact with the SNAPc-containing Pol II PIC described here and how a nucleosome may enable or modulate this interaction. ...
RNA polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes, such as small nuclear RNA (snRNA) genes, is less well understood at the structural level. Here, we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol IIpre-initiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE), located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc, called wing-1 and wing-2, bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.
... To our surprise, 7SKsnRNA, a well characterized short RNAs [35] strongly bound with lycopene at physiological conditions, throws light on the reversible autophagy observed in SMCs. Our observation was in great agreement with growing evidence on the short nc-RNAs regulating functions of cells through transcriptional regulation of survival genes [36]. ...
The chance of survival rate and autophagy of smooth muscle cells under calcium stress were drastically improved with a prolonged inclusion of Lycopene in the media. The results showed an improved viability from 41% to 69% and a reduction in overall autophagic bodies from 7% to 3%, which was well in agreement with the LC3II and III mRNA levels. However, the proliferation was slow compared to the controls. The fall in the major inflammatory marker TNF-α and improved antioxidant enzyme GPx were regarded as significant restoration markers of cell survival. The reactive oxygen species (ROS) were reduced from 8 fold to 3 fold post addition of lycopene for 24 h. Further, the docking studies revealed binding of lycopene molecules with 7SK snRNA at 7.6 kcal/mol docking energy with 300 ns stability under physiological conditions. Together, these results suggest that Lycopene administration during ischemic heart disease might improve the functions of the smooth muscle cells and 7SK snRNA might be involved in the binding of lycopene and its antioxidant protective effects.
... However, a new division was proposed, a midsize, noncoding RNAs that are 50-400 nucleotides long [6]. In this new category, multiple RNA types may be distinguished: transfer RNA (tRNA); small, nuclear RNA (snRNA); small, nucleolar RNA (snoRNA); small Cajal body-specific RNA (scaRNA) and YRNA [2,[7][8][9][10][11][12][13][14][15][16][17][18]. In this review, each of midsize noncoding RNAs will be analyzed in terms of their structure, biogenesis, function and influence on cancer in order to systematize current knowledge in the field. ...
... This process occurs in the cytoplasm and fully formed snRNPs are transferred back to the nucleus to perform one of their many functions. Genes for the major snRNAs are transcribed by Pol II and the most common are U1, U2, U4 and U5 molecules [10,53]. Interestingly, U6 spliceosomal RNAs, 7SK and 7SL, are transcribed by RNA Pol III [10,[54][55][56][57]. ...
... Genes for the major snRNAs are transcribed by Pol II and the most common are U1, U2, U4 and U5 molecules [10,53]. Interestingly, U6 spliceosomal RNAs, 7SK and 7SL, are transcribed by RNA Pol III [10,[54][55][56][57]. The snRNA genes have a less complex promoter in contrast to protein-coding ones. ...
Most of the human genome is made out of noncoding RNAs (ncRNAs). These ncRNAs do not code for proteins but carry a vast number of important functions in human cells such as: modification and processing other RNAs (tRNAs, rRNAs, snRNAs, snoRNAs, miRNAs), help in the synthesis of ribosome proteins, initiation of DNA replication, regulation of transcription, processing of pre-messenger mRNA during its maturation and much more. The ncRNAs also have a significant impact on many events that occur during carcinogenesis in cancer cells, such as: regulation of cell survival, cellular signaling, apoptosis, proliferation or even influencing the metastasis process. The ncRNAs may be divided based on their length, into short and long, where 200 nucleotides is the “magic” border. However, a new division was proposed, suggesting the creation of the additional group called midsize noncoding RNAs, with the length ranging from 50 – 400 nucleotides. This new group may include: transfer RNA (tRNA), small nuclear RNAs (snRNAs) with 7SK and 7SL, small nucleolar RNAs (snoRNAs), small Cajal body-specific RNAs (scaRNAs) and YRNAs. In this review their structure, biogenesis, function and influence on carcinogenesis process will be evaluated. What is more, a question will be answered of whether this new division is a necessity that clears current knowledge or just creates an additional misunderstanding in the ncRNA world?
... U snRNAs are short (100-200bp) uridinerich ncRNAs that have essential roles in premRNA splicing. The U snRNAs U1, U2, U4 and U5 are embedded in the spliceosome, whereas U3 and U7 are implicated in ribosomal RNA processing and transcription termination of histone mRNAs, respectively [50][51][52] . Integrator has emerged as the key U snRNA transcription termination machinery, mirroring the role of CPSF at proteincoding genes. ...
... Furthermore, an (A+T)rich sequence of ~14-16 nucleotides, termed the '3′ box' , is consistently found in human snRNA loci 57 . The 3′ box is essential for U1 ter mination, and is positioned immediately downstream of the processed 3′ end 52 . Upon depletion of either INTS11 or INTS1, lack of cleavage activity leads to accumulation of unprocessed transcripts spanning the 3′ box 21 . ...
In higher eukaryotes, fine-tuned activation of protein-coding genes and many non-coding RNAs pivots around the regulated activity of RNA polymerase II (Pol II). The Integrator complex is the only Pol II-associated large multiprotein complex that is metazoan specific, and has therefore been understudied for years. Integrator comprises at least 14 subunits, which are grouped into distinct functional modules. The phosphodiesterase activity of the core catalytic module is co-transcriptionally directed against several RNA species, including long non-coding RNAs (lncRNAs), U small nuclear RNAs (U snRNAs), PIWI-interacting RNAs (piRNAs), enhancer RNAs and nascent pre-mRNAs. Processing of non-coding RNAs by Integrator is essential for their biogenesis, and at protein-coding genes, Integrator is a key modulator of Pol II promoter-proximal pausing and transcript elongation. Recent studies have identified an Integrator-specific serine/threonine-protein phosphatase 2A (PP2A) module, which targets Pol II and other components of the basal transcription machinery. In this Review, we discuss how the activity of Integrator regulates transcription, RNA processing, chromatin landscape and DNA repair. We also discuss the diverse roles of Integrator in development and tumorigenesis. Integrator is the only metazoan-specific RNA polymerase II (Pol II)-associated large multisubunit complex. Processing of non-coding RNAs by Integrator is essential for their biogenesis, and, at protein-coding genes, Integrator regulates Pol II promoter-proximal pausing and elongation. Consequently, Integrator has diverse roles in development and tumorigenesis.
... Uridylation is thought to recognize misprocessed and/or low-quality snRNPs and target them for degradation (96,(101)(102)(103). The variation in the 3 ends of snRNAs may reflect differences in promoter sequences (104), gene bodies or 3 regions, which are crucial determinants for the cleavage and maturation of the snRNA precursor transcripts (105). The different susceptibilities of individual snRNAs to loss of maturation factors might also depend on the specificity of these factors in 3 -end processing and on their residual levels. ...
Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5′-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3′ tails that are often uridylated. snRNAs with defective 3′ terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.
... INTS14 is one of the subunits of the Integrator complex, which mediates the 3 -end processing of small nuclear RNA (snRNA). snRNA is a component of the spliceosome required for the splicing of pre-mRNA and for the expression of protein-coding genes [19][20][21][22][23] [26]. The knockdown of INTS14 in PC-3 resulted in a decrease in MYC mRNA and protein expression, as well as G0/1 arrest. ...
MYC is a major oncogene that plays an important role in cell proliferation in human cancers. Therefore, the mechanism behind MYC regulation is a viable therapeutic target for the treatment of cancer. Comprehensive and efficient screening of MYC regulators is needed, and we had previously established a promoter screening system using fluorescent proteins and the CRISPR library. For the efficient identification of candidate genes, a database was used, for which mRNA expression was correlated with MYC using datasets featuring "Similar" and "Not exactly similar" contexts. INTS14 and ERI2 were identified using datasets featuring the "Similar" context group, and INTS14 and ERI2 were capable of enhancing MYC promoter activity. In further database analysis of human cancers, a higher expression of MYC mRNA was observed in the INTS14 mRNA high-expressing prostate and liver cancers. The knockdown of INTS14 in prostate cell lines resulted in decreased MYC mRNA and protein expression and also induced G0/1 arrest. This study confirmed that CRISPR screening combined with context-matched database screening is effective in identifying genes that regulate the MYC promoter. This method can be applied to other genes and is expected to be useful in identifying the regulators of other proto-oncogenes.
