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 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 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 Identification of cis Elements Directing Termination of Yeast Nonpolyadenylated snoRNA Transcripts

2004 ◽  
Vol 24 (14) ◽  
pp. 6241-6252 ◽  
Author(s):  
Kristina L. Carroll ◽  
Dennis A. Pradhan ◽  
Josh A. Granek ◽  
Neil D. Clarke ◽  
Jeffry L. Corden
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Large Degree ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Gel Mobility Shift

ABSTRACT RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.

scholarly journals Direct Interactions between the Paf1 Complex and a Cleavage and Polyadenylation Factor Are Revealed by Dissociation of Paf1 from RNA Polymerase II

2008 ◽  
Vol 7 (7) ◽  
pp. 1158-1167 ◽  
Author(s):  
Kristen Nordick ◽  
Matthew G. Hoffman ◽  
Joan L. Betz ◽  
Judith A. Jaehning
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Direct Interaction ◽  
Pol Ii ◽  
Phosphorylated Form ◽  
Cleavage And Polyadenylation ◽  
Paf1 Complex ◽  
Processing Factors ◽  
Polyadenylation Factor ◽  
Transcription Cycle

ABSTRACT The Paf1 complex (Paf1, Ctr9, Cdc73, Rtf1, and Leo1) is normally associated with RNA polymerase II (Pol II) throughout the transcription cycle. However, the loss of either Rtf1 or Cdc73 results in the detachment of the Paf1 complex from Pol II and the chromatin form of actively transcribed genes. Using functionally tagged forms of the Paf1 complex factors, we have determined that, except for the more loosely associated Rtf1, the remaining components stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin. The loss of Paf1, Ctr9, or to a lesser extent Cdc73 or Rtf1 results in reduced levels of serine 2 phosphorylation of the Pol II C-terminal domain and in increased read through of the MAK21 polyadenylation site. We found that the cleavage and polyadenylation factor Cft1 requires the Pol II-associated form of the Paf1 complex for full levels of interaction with the serine 5-phosphorylated form of Pol II. When the Paf1 complex is dissociated from Pol II, a direct interaction between Cft1 and the Paf1 complex can be detected. These results are consistent with the Paf1 complex providing a point of contact for recruitment of 3′-end processing factors at an early point in the transcription cycle. The lack of this connection helps to explain the defects in 3′-end formation observed in the absence of Paf1.

scholarly journals The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation

2021 ◽  
Author(s):  
Yavor Hadzhiev ◽  
Lucy Wheatley ◽  
Ledean Cooper ◽  
Federico Ansaloni ◽  
Celina Whalley ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Gene Cluster ◽  
Core Promoter ◽  
Single Copy ◽  
Early Embryo ◽  
Zygotic Genome Activation ◽  
Pol Ii ◽  
A Minor ◽  
Genome Activation ◽  
Zygotic Genome

In anamniote embryos the major wave of zygotic genome activation (ZGA) starts during the mid-blastula transition. This major wave of ZGA is facilitated by several mechanisms, including dilution of repressive maternal factors and accumulation of activating transcription factors during the fast cell division cycles preceding the mid-blastula transition. However, a set of genes escape global genome repression and are activated substantially earlier, during what is called, the minor wave of genome activation. While the mechanisms underlying the major wave of genome activation have been studied extensively, the minor wave of genome activation is little understood. In zebrafish the earliest expressed RNA polymerase II (Pol II) transcribed genes are activated in a pair of large transcription bodies depleted of chromatin, abundant in elongating Pol II and nascent RNAs (Hadzhiev et al., 2019; Hilbert et al., 2021). This transcription body includes the miR-430 gene cluster required for maternal mRNA clearance. Here we explored the genomic, chromatin organisation and cis-regulatory mechanisms of the minor wave of genome activation occurring in the transcription body. By long read genome sequencing we identified a remarkable cluster of miR-430 genes with over 300 promoters and spanning 0.6 Mb, which represent the highest promoter density of the genome. We demonstrate that the miR-430 gene cluster is required for the formation of the transcription body and acts as a transcription organiser for minor wave activation of a set of zinc finger genes scattered on the same chromosome arm, which share promoter features with the miR-430 cluster. These promoter features are shared among minor wave genes overall and include the TATA-box and sharp transcription start site profile. Single copy miR-430 promoter transgene reporter experiments indicate the importance of promoter-autonomous mechanisms regulating escape from global repression of the early embryo. These results together suggest that formation of the transcription body in the early embryo is the result of high promoter density coupled to a minor wave-specific core promoter code for transcribing key minor wave ZGA genes, which are required for the overhaul of the transcriptome during early embryonic development.

scholarly journals Transcription and splicing dynamics during early Drosophila development

2020 ◽  
Author(s):  
Pedro Prudêncio ◽  
Kenny Rebelo ◽  
Rosina Savisaar ◽  
Rui Gonçalo Martinho ◽  
Maria Carmo-Fonseca
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Dynamic Features ◽  
Antisense Gene ◽  
Pol Ii ◽  
Human Genes ◽  
Yeast Genes ◽  
Splice Junctions ◽  
Kinetics Of ◽  
Intron Definition

ABSTRACTWidespread co-transcriptional splicing has been demonstrated from yeast to human. However, measuring the kinetics of splicing relative to transcription has been hampered by technical challenges. Here, we took advantage of native elongating transcript sequencing (NET-seq) to identify the position of RNA polymerase II (Pol II) when exons become ligated in the newly synthesized RNA. We analyzed Drosophila melanogaster embryos because the genes transcribed initially during development have few and short introns (like yeast genes), whereas genes transcribed later contain multiple long introns (more similar to human genes). We detected spliced NET-seq reads connected to Pol II molecules that were positioned just a few nucleotides downstream of the 3’ splice site. Although the majority of splice junctions were covered by spliced reads, many introns remained unspliced, resulting in a complex range of heterogeneity in splicing dynamics. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. Both canonical and recursive splicing were associated with a higher Pol II density, suggesting a splicing-coupled mechanism that slows down transcription elongation. We further observed that transcription termination was very efficient for isolated genes but that the presence of an overlapping antisense gene was often associated with transcriptional read-through. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

scholarly journals An "individualist" model of an active genome in a developing embryo

2021 ◽  
Author(s):  
Shao-Kuei Huang ◽  
Sayantan Dutta ◽  
Peter H Whitney ◽  
Stanislav Shvartsman ◽  
Christine Rushlow
Keyword(s):  
Rna Polymerase Ii ◽  
Physical Distance ◽  
Drosophila Embryo ◽  
Drosophila Genome ◽  
Pol Ii ◽  
Individual Gene ◽  
Early Drosophila Embryo ◽  
Primary Driver ◽  
Genome Activation ◽  
Active Genes

The early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the whole genome. Using Drosophila embryos undergoing the first wave of genome activation, we detected discrete "speckles" of RNA Polymerase II (Pol II), and showed that they overlap with transcribing loci. We characterized the spatial distribution of Pol II speckles and quantified how this distribution changes in the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda. Although the number and size of Pol II speckles were reduced, indicating that Zelda promotes Pol II speckle formation, we observed a uniform distribution of distances between active genes in the nuclei of both wildtype and zelda mutant embryos. This suggests that the topologically associated domains identified by Hi-C studies do little to spatially constrain groups of transcribed genes at this time. We provide evidence that linear genomic distance between transcribed genes is the primary determinant of measured physical distance between the active loci. Furthermore, we show active genes can have distinct Pol II pools even if the active loci are in close proximity. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, our data support an "individualist" model of gene control at early genome activation in Drosophila. This model is in contrast to a "collectivist" model where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems.

scholarly journals A Cdk9-PP1 kinase-phosphatase switch regulates the elongation-termination transition of RNA polymerase II

2017 ◽  
Author(s):  
Pabitra K. Parua ◽  
Gregory T. Booth ◽  
Miriam Sansó ◽  
Bradley Benjamin ◽  
Jason C. Tanny ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Bistable Switch ◽  
Pol Ii ◽  
Mrna Maturation ◽  
Cleavage And Polyadenylation ◽  
Transcription Complexes

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to ensure proper mRNA maturation and prevent interference between neighboring genes1. Pol II slowing downstream of the cleavage and polyadenylation signal (CPS) leads to recruitment of cleavage and polyadenylation factors and termination2, but how this chain of events is initiated remains unclear. In a chemical-genetic screen, we identified protein phosphatase 1 (PP1) isoforms as substrates of human positive transcription elongation factor b (P-TEFb), the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 complex3. Here we show that Cdk9 and PP1 govern phosphorylation of the conserved transcription factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis24. Sites phosphorylated by Cdk9 in the Spt5 carboxy-terminal domain (CTD) are dephosphorylated by Dis2 in vitro, and Cdk9 inhibition in vivo leads to rapid Spt5 dephosphorylation that is retarded by concurrent Dis2 inactivation. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, prior to or concomitant with slowing of Pol II5. A Dis2-inactivating mutation stabilizes Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq)6, and is suppressed by ablation of Cdk9 target sites in Spt5. These results support a model whereby the transition of Pol II from elongation to termination is regulated by opposing activities of Cdk9 and Dis2 towards their common substrate Spt5—a bistable switch analogous to a Cdk1-PP1 module that controls mitotic progression4.

scholarly journals Recruitment of the putative transcription-repair coupling factor CSB/ERCC6 to RNA polymerase II elongation complexes.

1997 ◽  
Vol 17 (12) ◽  
pp. 6803-6814 ◽  
Author(s):  
D Tantin ◽  
A Kansal ◽  
M Carey
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Excision Repair ◽  
Coupling Factor ◽  
Pol Ii ◽  
Growth Defects ◽  
Cockayne's Syndrome ◽  
Nucleotide Excision ◽  
Rna Complex ◽  
Dna Template

Cockayne's syndrome (CS) is a disease characterized by developmental and growth defects, sunlight sensitivity, and a defect in transcription-coupled nucleotide excision repair. The two principle proteins involved in CS, CSA and CSB/ERCC6, have been hypothesized to bind RNA polymerase II (Pol II) and link transcription to DNA repair. We have tested CSA and CSB in assays designed to determine their role in transcription-coupled repair. Using a unique oligo(dC)-tailed DNA template, we provide biochemical evidence that CSB/ERCC6 interacts with Pol II molecules engaged in ternary complexes containing DNA and nascent RNA. CSB is a DNA-activated ATPase, and hydrolysis of the ATP beta-gamma phosphoanhydride bond is required for the formation of a stable Pol II-CSB-DNA-RNA complex. Unlike CSB, CSA does not directly bind Pol II.

scholarly journals Association of SARFH (sarcoma-associated RNA-binding fly homolog) with regions of chromatin transcribed by RNA polymerase II.

1995 ◽  
Vol 15 (8) ◽  
pp. 4562-4571 ◽  
Author(s):  
D Immanuel ◽  
H Zinszner ◽  
D Ron
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Polytene Chromosomes ◽  
Drosophila Embryo ◽  
Cdna Clones ◽  
Rna Pol Ii ◽  
Pol Ii

Many oncogenes associated with human sarcomas are composed of a fusion between transcription factors and the N-terminal portions of two similar RNA-binding proteins, TLS and EWS. Though the oncogenic fusion proteins lack the RNA-binding domain and do not bind RNA, the contribution from the N-terminal portion of the RNA-binding protein is essential for their transforming activity. TLS and EWS associate in vivo with RNA polymerase II (Pol II) transcripts. To learn more about the target gene specificity of this interaction, the localization of a Drosophila melanogaster protein that has extensive sequence identity to the C-terminal RNA-binding portions of TLS and EWS was studied in preparations of Drosophila polytene nuclei. cDNA clones encoding the full-length Drosophila TLS-EWS homolog, SARFH (stands for sarcoma-associated RNA-binding fly homolog), were isolated. Functional similarity to TLS and EWS was revealed by the association of SARFH with Pol II transcripts in mammalian cells and by the ability of SARFH to elicit homologous down-regulation of the levels of the mammalian proteins. The SARFH gene is expressed in the developing Drosophila embryo from the earliest stages of cellularization and is subsequently found in many cell types. In preparations of polytene chromosomes from salivary gland nuclei, SARFH antibodies recognize their target associated with the majority of active transcription units, revealed by colocalization with the phosphorylated form of RNA Pol II. We conclude that SARFH and, by homology, EWS and TLS participate in a function common to the expression of most genes transcribed by RNA Pol II.

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