scholarly journals Transcript isoform sequencing reveals widespread promoter-proximal transcriptional termination

2019 ◽  
Author(s):  
Ryan Ard ◽  
Quentin Thomas ◽  
Bingnan Li ◽  
Jingwen Wang ◽  
Vicent Pelechano ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nascent Transcript ◽  
Transcription Start Sites ◽  
Transcriptional Termination ◽  
Isoform Diversity ◽  
Transcript Cleavage ◽  
Cleavage And Polyadenylation ◽  
Gene Isoform ◽  
Alternative Tsss

SUMMARYHigher organisms achieve optimal gene expression by tightly regulating the transcriptional activity of RNA Polymerase II (RNAPII) along DNA sequences of genes1. RNAPII density across genomes is typically highest where two key choices for transcription occur: near transcription start sites (TSSs) and polyadenylation sites (PASs) at the beginning and end of genes, respectively2,3. Alternative TSSs and PASs amplify the number of transcript isoforms from genes4, but how alternative TSSs connect to variable PASs is unresolved from common transcriptomics methods. Here, we define TSS/PAS pairs for individual transcripts in Arabidopsis thaliana using an improved Transcript Isoform sequencing (TIF-seq) protocol and find on average over four different isoforms corresponding to variable TSS/PAS pairs per expressed gene. While intragenic initiation represents a large source of regulated isoform diversity, we discover that ∼ 14% of expressed genes generate relatively unstable short promoter-proximal RNAs (sppRNAs) from nascent transcript cleavage and polyadenylation shortly after initiation. The location of sppRNAs coincides with increased RNAPII density, indicating these large pools of promoter-stalled RNAPII across genomes are often engaged in transcriptional termination. RNAPII elongation factors progress transcription beyond sites of sppRNA formation, demonstrating RNAPII density near promoters represents a checkpoint for early transcriptional termination that governs full-length gene isoform expression.

scholarly journals AT-rich sequence elements promote nascent transcript cleavage leading to RNA polymerase II termination

2012 ◽  
Vol 41 (3) ◽  
pp. 1797-1806 ◽  
Author(s):  
Eleanor White ◽  
Kinga Kamieniarz-Gdula ◽  
Michael J. Dye ◽  
Nick J. Proudfoot
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nascent Transcript ◽  
Transcript Cleavage ◽  
Sequence Elements

scholarly journals cis- and trans-Acting Determinants of Transcription Termination by Yeast RNA Polymerase II

2006 ◽  
Vol 26 (7) ◽  
pp. 2688-2696 ◽  
Author(s):  
Eric J. Steinmetz ◽  
Sarah B. H. Ng ◽  
Joseph P. Cloute ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Nascent Transcript ◽  
Protein Coding ◽  
Pol Ii ◽  
Eukaryotic Genes ◽  
Discrete Surface ◽  
Cleavage And Polyadenylation ◽  
Selection For

ABSTRACT Most eukaryotic genes are transcribed by RNA polymerase II (Pol II), including those that produce mRNAs and many noncoding functional RNAs. Proper expression of these genes requires efficient termination by Pol II to avoid transcriptional interference and synthesis of extended, nonfunctional RNAs. We previously described a pathway for yeast Pol II termination that involves recognition of an element in the nascent transcript by the essential RNA-binding protein Nrd1. The Nrd1-dependent pathway appears to be used primarily for nonpolyadenylated transcripts, such as the small nuclear and small nucleolar RNAs (snoRNAs). mRNAs are thought to use a distinct pathway that is coupled to cleavage and polyadenylation of the transcript. Here we show that the terminator elements for two yeast snoRNA genes also direct polyadenylated 3′-end formation in the context of an mRNA 3′ untranslated region. A selection for cis-acting terminator readthrough mutations identified conserved features of these elements, some of which are similar to cleavage and polyadenylation signals. A selection for trans-acting mutations that induce readthrough of both a snoRNA and an mRNA terminator yielded mutations in the Rpb3 and Rpb11 subunits of Pol II that define a remarkably discrete surface on the trailing end of the enzyme. Our results suggest that, at least in budding yeast, protein-coding and noncoding Pol II-transcribed genes use similar mechanisms to direct termination and that the termination signal is transduced through the Rpb3/Rpb11 heterodimer.

scholarly journals Distinct nucleosome distribution patterns in two structurally and functionally differentiated nuclei of a unicellular eukaryote

2015 ◽  
Author(s):  
Jie Xiong ◽  
Shan Gao ◽  
Wen Dui ◽  
Wentao Yang ◽  
Xiao Chen ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Germ Line ◽  
Nucleosome Occupancy ◽  
Gc Content ◽  
Distribution Patterns ◽  
Unicellular Eukaryote ◽  
Transcription Start Sites ◽  
Highly Correlated ◽  
Cis And Trans

The ciliate protozoan Tetrahymena thermophila contains two types of structurally and functionally differentiated nuclei: the transcriptionally active somatic macronucleus (MAC) and the transcriptionally silent germ-line micronucleus (MIC). Here we demonstrate that MAC features well-positioned nucleosomes downstream of transcription start sites (TSS) likely connected with promoter proximal pausing of RNA polymerase II, as well as in exonic regions flanking both the 5′ and 3′ splice sites. In contrast, nucleosomes in MIC are more delocalized. Nucleosome occupancy in MAC and MIC are nonetheless highly correlated with each other and with predictions based upon DNA sequence features. Arrays of well-positioned nucleosomes are often correlated with GC content oscillations, suggesting significant contributions from cis-determinants. We propose that cis- and trans-determinants may coordinately accommodate some well-positioned nucleosomes with important functions, driven by a process in which positioned nucleosomes shape the mutational landscape of associated DNA sequences, while the DNA sequences in turn reinforce nucleosome positioning.

scholarly journals Transcription Factors TFIIF, ELL, and Elongin Negatively Regulate SII-induced Nascent Transcript Cleavage by Non-arrested RNA Polymerase II Elongation Intermediates

2001 ◽  
Vol 276 (25) ◽  
pp. 23109-23114 ◽  
Author(s):  
B. Jean Elmendorf ◽  
A. Shilatifard ◽  
Qin Yan ◽  
Joan Weliky Conaway ◽  
Ronald C. Conaway
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nascent Transcript ◽  
Transcript Cleavage

scholarly journals NELF and GAGA Factor Are Linked to Promoter-Proximal Pausing at Many Genes in Drosophila

2008 ◽  
Vol 28 (10) ◽  
pp. 3290-3300 ◽  
Author(s):  
Chanhyo Lee ◽  
Xiaoyong Li ◽  
Aaron Hechmer ◽  
Michael Eisen ◽  
Mark D. Biggin ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Proximal Region ◽  
Gaga Factor ◽  
Pol Ii ◽  
Transcription Start Sites ◽  
Genome Wide ◽  
Highly Expressed Genes ◽  
Inactive Genes ◽  
Chip Chip

ABSTRACT Recent analyses of RNA polymerase II (Pol II) revealed that Pol II is concentrated at the promoters of many active and inactive genes. NELF causes Pol II to pause in the promoter-proximal region of the hsp70 gene in Drosophila melanogaster. In this study, genome-wide location analysis (chromatin immunoprecipitation-microarray chip [ChIP-chip] analysis) revealed that NELF is concentrated at the 5′ ends of 2,111 genes in Drosophila cells. Permanganate genomic footprinting was used to determine if paused Pol II colocalized with NELF. Forty-six of 56 genes with NELF were found to have paused Pol II. Pol II pauses 30 to 50 nucleotides downstream from transcription start sites. Analysis of DNA sequences in the vicinity of paused Pol II identified a conserved DNA sequence that probably associates with TFIID but detected no evidence of RNA secondary structures or other conserved sequences that might directly control elongation. ChIP-chip experiments indicate that GAGA factor associates with 39% of the genes that have NELF. Surprisingly, NELF associates with almost one-half of the most highly expressed genes, indicating that NELF is not necessarily a repressor of gene expression. NELF-associated pausing of Pol II might be an obligatory but sometimes transient checkpoint during the transcription cycle.

scholarly journals Transcription and splicing dynamics during early Drosophila development

RNA ◽  
2021 ◽  
pp. rna.078933.121
Author(s):  
Pedro Prudencio ◽  
Rosina Savisaar ◽  
Kenny Rebelo ◽  
Rui Goncalo Martinho ◽  
Maria Carmo-Fonseca
Keyword(s):  
Rna Polymerase Ii ◽  
Developmental Stages ◽  
Early Embryo ◽  
Drosophila Embryo ◽  
Dynamic Features ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Cleavage And Polyadenylation ◽  
Dna Template ◽  
Early Developmental

Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nucleotides of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

scholarly journals Ssu72 Protein Mediates Both Poly(A)-Coupled and Poly(A)-Independent Termination of RNA Polymerase II Transcription

2003 ◽  
Vol 23 (18) ◽  
pp. 6339-6349 ◽  
Author(s):  
Eric J. Steinmetz ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genetic Selection ◽  
Common Mechanism ◽  
Snorna Gene ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Gene Coding ◽  
Eukaryotic Gene ◽  
Cleavage And Polyadenylation

ABSTRACT Termination of transcription by RNA polymerase II (Pol II) is a poorly understood yet essential step in eukaryotic gene expression. Termination of pre-mRNA synthesis is coupled to recognition of RNA signals that direct cleavage and polyadenylation of the nascent transcript. Termination of nonpolyadenylated transcripts made by Pol II in the yeast Saccharomyces cerevisiae, including the small nuclear and small nucleolar RNAs, requires distinct RNA elements recognized by the Nrd1 protein and other factors. We have used genetic selection to characterize the terminator of the SNR13 snoRNA gene, revealing a bipartite structure consisting of an upstream element closely matching a Nrd1-binding sequence and a downstream element similar to a cleavage/polyadenylation signal. Genome-wide selection for factors influencing recogniton of the SNR13 terminator yielded mutations in the gene coding for the essential Pol II-binding protein Ssu72. Ssu72 has recently been found to associate with the pre-mRNA cleavage/polyadenylation machinery, and we find that an ssu72 mutation that disrupts Nrd1-dependent termination also results in deficient poly(A)-dependent termination. These findings extend the parallels between the two termination pathways and suggest that they share a common mechanism to signal Pol II termination.

scholarly journals Transcription-driven Chromatin Repression of Intragenic Promoters

2018 ◽  
Author(s):  
Mathias Nielsen ◽  
Ryan Ard ◽  
Xueyuan Leng ◽  
Maxim Ivanov ◽  
Peter Kindgren ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Positional Information ◽  
Gene Promoters ◽  
Molecular Signatures ◽  
Transcription Start Sites ◽  
Histone 3 ◽  
Isoform Diversity ◽  
Genome Wide ◽  
Chaperone Complex ◽  
Rnapii Transcription

SummaryProgression of RNA polymerase II (RNAPII) transcription relies on the appropriately positioned activities of elongation factors. The resulting profile of factors and chromatin signatures along transcription units provides a “positional information system” for transcribing RNAPII. Here, we investigate a chromatin-based mechanism that suppresses intragenic initiation of RNAPII transcription. We demonstrate that RNAPII transcription across gene promoters represses their function in plants. This repression is characterized by reduced promoter-specific molecular signatures and increased molecular signatures associated with RNAPII elongation. The FACT histone chaperone complex is required for this repression mechanism. Genome-wide mapping of Transcription Start Sites (TSSs) reveals thousands of discrete intragenic TSS positions in FACT mutants. Histone 3 lysine 4 mono-methylation poises exonic sites to initiate RNAPII transcription in FACT mutants. Uncovering the mechanism for intragenic TSS repression through the act of RNAPII elongation has important implications for understanding pervasive RNAPII transcription and the regulation of transcript isoform diversity.

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