scholarly journals Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq

2021 ◽  
pp. gr.275784.121
Author(s):  
Bo Yan ◽  
George Tzertzinis ◽  
Ira Schildkraut ◽  
Laurence Ettwiller
Keyword(s):  
Noncoding Rna ◽  
Rna Polymerases ◽  
Transcription Start ◽  
Pol Ii ◽  
Transcription Start Sites ◽  
Cell Functions ◽  
Wide Scale ◽  
Nucleotide Resolution ◽  
Pol Iii

Determination of eukaryotic Transcription Start Sites (TSS) has been based on methods that require the cap structure at the 5-prime end of transcripts derived from Pol-II RNA polymerase. Consequently, these methods do not reveal TSS derived from the other RNA polymerases which also play critical roles in various cell functions. To address this limitation, we developed ReCappable-seq which comprehensively identifies TSS for both Pol-lI and non-Pol-II transcripts at single-nucleotide resolution. The method relies on specific enzymatic exchange of 5-prime m7G caps and 5-prime triphosphates with a selectable tag. When applied to human transcriptomes, ReCappable-seq identifies Pol-II TSS that are in agreement with orthogonal methods such as CAGE. Additionally, ReCappable-seq reveals a rich landscape of TSS associated with Pol-III transcripts which have not previously been amenable to study at genome-wide scale. Novel TSS from non-Pol-II transcription can be located in the nuclear and mitochondrial genomes. ReCappable-seq interrogates the regulatory landscape of coding and noncoding RNA concurrently and enables the classification of epigenetic profiles associated with Pol-lI and non-Pol-II TSS.

scholarly journals ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases

2019 ◽  
Author(s):  
Bo Yan ◽  
George Tzertzinis ◽  
Ira Schildkraut ◽  
Laurence Ettwiller
Keyword(s):  
Rna Polymerases ◽  
Transcription Start ◽  
Eukaryotic Transcription ◽  
Pol Ii ◽  
Transcription Start Sites ◽  
Cell Functions ◽  
A Genome ◽  
Wide Scale ◽  
Nucleotide Resolution ◽  
Pol Iii

AbstractMethodologies for determining eukaryotic Transcription Start Sites (TSS) rely on the selection of the 5’ canonical cap structure of Pol-II transcripts and are consequently ignoring entire classes of TSS derived from other RNA polymerases which play critical roles in various cell functions. To overcome this limitation, we developed ReCappable-seq and identified TSS from Pol-ll and non-Pol-II transcripts at nucleotide resolution. Applied to the human transcriptome, ReCappable-seq identifies Pol-II TSS with higher specificity than CAGE and reveals a rich landscape of TSS associated notably with Pol-III transcripts which have been previously not possible to study on a genome-wide scale. Novel TSS consistent with non-Pol-II transcripts can be found in the nuclear and mitochondrial genomes. By identifying TSS derived from all RNA-polymerases, ReCappable-seq reveals distinct epigenetic marks among Pol-lI and non-Pol-II TSS and provides a unique opportunity to concurrently interrogate the regulatory landscape of coding and non-coding RNA.

scholarly journals Pol II and its associated epigenetic marks are present at Pol III–transcribed noncoding RNA genes

2010 ◽  
Vol 17 (5) ◽  
pp. 629-634 ◽  
Author(s):  
Artem Barski ◽  
Iouri Chepelev ◽  
Dritan Liko ◽  
Suresh Cuddapah ◽  
Alastair B Fleming ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Pol Ii ◽  
Epigenetic Marks ◽  
Pol Iii ◽  
Rna Genes

primirTSS: an R package for identifying cell-specific microRNA transcription start sites

2020 ◽  
Vol 36 (11) ◽  
pp. 3605-3606
Author(s):  
Pumin Li ◽  
Qi Xu ◽  
Xu Hua ◽  
Zhongwei Xie ◽  
Jie Li ◽  
...  
Keyword(s):  
Conservation Score ◽  
R Package ◽  
Supplementary Information ◽  
Transcription Start ◽  
Pol Ii ◽  
Transcription Start Sites ◽  
Web Interfaces ◽  
Multiple Datasets ◽  
R Programming ◽  
Programming Interfaces

Abstract Summary The R/Bioconductor package primirTSS is a fast and convenient tool that allows implementation of the analytical method to identify transcription start sites of microRNAs by integrating ChIP-seq data of H3K4me3 and Pol II. It further ensures the precision by employing the conservation score and sequence features. The tool showed a good performance when using H3K4me3 or Pol II Chip-seq data alone as input, which brings convenience to applications where multiple datasets are hard to acquire. This flexible package is provided with both R-programming interfaces as well as graphical web interfaces. Availability and implementation primirTSS is available at: http://bioconductor.org/packages/primirTSS. The documentation of the package including an accompanying tutorial was deposited at: https://bioconductor.org/packages/release/bioc/vignettes/primirTSS/inst/doc/primirTSS.html. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Pausing sites of RNA polymerase II on actively transcribed genes are enriched in DNA double-stranded breaks

2020 ◽  
Vol 295 (12) ◽  
pp. 3990-4000 ◽  
Author(s):  
Sandeep Singh ◽  
Karol Szlachta ◽  
Arkadi Manukyan ◽  
Heather M. Raimer ◽  
Manikarna Dinda ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Topoisomerase I ◽  
Cell Types ◽  
Dna Breaks ◽  
Transcription Start ◽  
Pol Ii ◽  
Transcription Start Sites

DNA double-stranded breaks (DSBs) are strongly associated with active transcription, and promoter-proximal pausing of RNA polymerase II (Pol II) is a critical step in transcriptional regulation. Mapping the distribution of DSBs along actively expressed genes and identifying the location of DSBs relative to pausing sites can provide mechanistic insights into transcriptional regulation. Using genome-wide DNA break mapping/sequencing techniques at single-nucleotide resolution in human cells, we found that DSBs are preferentially located around transcription start sites of highly transcribed and paused genes and that Pol II promoter-proximal pausing sites are enriched in DSBs. We observed that DSB frequency at pausing sites increases as the strength of pausing increases, regardless of whether the pausing sites are near or far from annotated transcription start sites. Inhibition of topoisomerase I and II by camptothecin and etoposide treatment, respectively, increased DSBs at the pausing sites as the concentrations of drugs increased, demonstrating the involvement of topoisomerases in DSB generation at the pausing sites. DNA breaks generated by topoisomerases are short-lived because of the religation activity of these enzymes, which these drugs inhibit; therefore, the observation of increased DSBs with increasing drug doses at pausing sites indicated active recruitment of topoisomerases to these sites. Furthermore, the enrichment and locations of DSBs at pausing sites were shared among different cell types, suggesting that Pol II promoter-proximal pausing is a common regulatory mechanism. Our findings support a model in which topoisomerases participate in Pol II promoter-proximal pausing and indicated that DSBs at pausing sites contribute to transcriptional activation.

The phylogenetic relations of DNA-dependent RNA polymerases of archaebacteria, eukaryotes, and eubacteria

1989 ◽  
Vol 35 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Wolfram Zillig ◽  
Hans-Peter Klenk ◽  
Peter Palm ◽  
Gabriela Pühler ◽  
Felix Gropp ◽  
...  
Keyword(s):  
Nuclear Genome ◽  
Distance Matrix ◽  
Gene Organization ◽  
Amino Acid Sequences ◽  
Rna Polymerases ◽  
Matrix Analysis ◽  
Pol Ii ◽  
Phylogenetic Relations ◽  
Pol I ◽  
Pol Iii

Unrooted phylogenetic dendrograms were calculated by two independent methods, parsimony and distance matrix analysis, from an alignment of the derived amino acid sequences of the A and C subunits of the DNA-dependent RNA polymerases of the archaebacteria Sulfolobus acidocaldarius and Halobacterium halobium with 12 corresponding sequences including a further set of archaebacterial A + C subunits, eukaryotic nuclear RNA polymerases, pol I, pol II, and pol III, eubacterial β′ and chloroplast β′ and β″ subunits. They show the archaebacteria as a coherent group in close neighborhood of and sharing a bifurcation with eukaryotic pol II and (or) pol IIIA components. The most probable trees show pol IA branching off from the tree separately at a bifurcation with the eubacterial β′ lineage. The implications of these results, especially for understanding the possibly chimeric origin of the eukaryotic nuclear genome, are discussed.Key words: transcription, evolution, taxonomy, subunits, gene organization.

scholarly journals Ssl2/TFIIH function in transcription start site scanning by RNA Polymerase II in Saccharomyces cerevisiae

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Tingting Zhao ◽  
Irina O Vvedenskaya ◽  
William KM Lai ◽  
Shrabani Basu ◽  
B Franklin Pugh ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Core Promoter ◽  
Interaction Network ◽  
Transcription Start ◽  
Pol Ii ◽  
Transcription Start Sites ◽  
Scanning Rate ◽  
Residue Interaction

In Saccharomyces cerevisiae, RNA Polymerase II (Pol II) selects transcription start sites (TSS) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window ~40-120 basepairs downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.

scholarly journals Structure of the TFIIIC subcomplex τA provides insights into RNA polymerase III pre-initiation complex formation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Matthias K. Vorländer ◽  
Anna Jungblut ◽  
Kai Karius ◽  
Florence Baudin ◽  
Helga Grötsch ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Dual Function ◽  
Start Site ◽  
Transcription Start ◽  
Initiation Complex ◽  
Pol Ii ◽  
Negative Stain ◽  
Pol Iii

Abstract Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.

scholarly journals Analysis of the Caenorhabditis elegans rpc-1 gene

2005 ◽  
Author(s):  
◽  
Qun Zheng
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Rna Polymerase Iii ◽  
Transgenic Animals ◽  
Rna Polymerases ◽  
Pol Ii ◽  
C Elegans ◽  
Pol I ◽  
Genes Encoding ◽  
Pol Iii

In eukaryotes, two large subunits form the core catalytic structure of RNA polymerase III (Pol III), which is conserved in other RNA polymerases, Pol I and Pol II. It has been found that Pol III activity is tightly associated to cell growth. TFIII B has been shown to be one of main mediators in this process. No regulation of the Pol III largest subunit gene has been found. In C. elegans, the rpc-1 gene encodes the largest subunit of Pol III. Here, I identified two critical structural components of RPC-1, Gly644 and Gly1055, whose mutations result in larval lethal arrestment. These two amino acid residues are universally conserved in RNA polymerases, indicating their overall involvement in gene transcription mechanism. Also, I found that maternally inherited, not embryonically expressed, rpc-1 gene products survive early development. Starvation was found to suppress rpc-1 gene expression and re-feeding treatment enhances rpc-1 gene expression rapidly. No similar regulation was detected in genes encoding largest subunits of Pol I and Pol II. This is the first time that rpc-1 gene regulation has been reported. Insulin signaling may not be involved in this regulation. Also, I found that rpc-1 promoter is not ubiquitously active in C. elegans. Using the rpc-1p::gfp transgene, the rpc-1 promoter activity is only detected in a subset of neurons in the head and the tail and the intestine. While starvation silences the rpc-1 promoter activity in most tissues and cells, ASK neurons still show GFP staining in the rpc-1p::gfp transgenic animals, indicating that rpc-1 transcription in ASK neurons is continuously active under starvation conditions. Further studies suggest that TGF-[beta] signaling is involved in mediating the rpc-1 promoter activity in ASK neurons.

scholarly journals Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II and Pol III

2020 ◽  
Author(s):  
Yongpeng Jiang ◽  
Jie Huang ◽  
Kehuan Lun ◽  
Boyuan Li ◽  
Haonan Zheng ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Chromatin Accessibility ◽  
Rna Polymerases ◽  
Chromatin Interaction ◽  
Transcription Inhibition ◽  
Pol Ii ◽  
3D Genome ◽  
Chromatin Interactions ◽  
Pol I ◽  
Pol Iii

AbstractBackgroundThe relationship between transcription and the 3D genome organization is one of the most important questions in molecular biology, but the roles of transcription in 3D chromatin remain controversial. Multiple groups showed that transcription affects global Cohesin binding and genome 3D structures. At the same time, several studies have indicated that transcription inhibition does not affect global chromatin interactions.ResultsHere, we provide the most comprehensive evidence to date to demonstrate that transcription plays a marginal role in organizing the 3D genome in mammalian cells: 1) degraded Pol I, Pol II and Pol III proteins in mESCs, and showed their loss results in little or no changes of global 3D chromatin structures for the first time; 2) selected RNA polymerases high abundance binding sites-associated interactions and found they still persist after the degradation; 3) generated higher resolution chromatin interaction maps and revealed that transcription inhibition mildly alters small loop domains; 4) identified Pol II bound but CTCF and Cohesin unbound loops and disclosed that they are largely resistant to transcription inhibition. Interestingly, Pol II depletion for a longer time significantly affects the chromatin accessibility and Cohesin occupancy, suggesting RNA polymerases are capable of affecting the 3D genome indirectly. So, the direct and indirect effects of transcription inhibition explain the previous confusing effects on the 3D genome.ConclusionsWe conclude that Pol I, Pol II, and Pol III loss only mildly alter chromatin interactions in mammalian cells, suggesting the 3D chromatin structures are pre-established and relatively stable.

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