STAR Protocols, Год журнала: 2024, Номер 5(4), С. 103369 - 103369
Опубликована: Окт. 10, 2024
Язык: Английский
PLoS ONE, Год журнала: 2025, Номер 20(6), С. e0321120 - e0321120
Опубликована: Июнь 2, 2025
The DEDD family of exonucleases has expanded through evolution whilst retaining a conserved catalytic domain. One subgroup with closely related domain sequences includes the yeast enzymes Rex1 (RNA exonuclease 1) and Rex3, metazoan REXO1 1 homologue) Rexo5 proteins, plant protein Sdn5 (small RNA degrading nuclease). Comparison structure models sequence analyses revealed that this group can be differentiated into two distinct clades consisting Rex1, on one hand, Rex3 other. Rex1-related proteins is inserted within conserved, discontinuous alkaline phosphatase (AlkP) AlkP contains three surface loops are modelled to directed towards domain, which forms an extended helical arch found in homologues across fungi plants. We show adjacent loop required for Rex1-mediated processing 5S rRNA tRNA Saccharomyces cerevisiae . Rex3-related including REXO1, lack but contain KIX (CREB kinase-inducible (KID) interacting domain) cysteine- histidine-rich (CHORD) C-terminal Deletion N-terminal region spanning blocked its function RNase MRP processing. In contrast metazoans not plants or algae. This work identifies evolutionarily structural hallmarks demonstrates specific features Rex1- Rex3-mediated pathways vivo.
Язык: Английский
Процитировано
0
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown
Опубликована: Ноя. 30, 2023
Abstract The ring-shaped cohesin complex topologically entraps two DNAs to establish sister chromatid cohesion 1–3 . Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation 4–7 , which is thought achieve by actively extruding DNA loops without entrapping 8–11 ‘loop extrusion’ hypothesis finds motivation from in vitro observations 12–14 – whether this process underlies vivo loop formation remains untested. Here, using budding yeast S. cerevisiae we generate variants that have lost their ability extrude but retain entrap DNA. Analysis of these suggests form independently extrusion. Instead, find transcription promotes formation, as well acts an extrinsic motor expands and defines ultimate positions. Our results necessitate a re-evaluation extrusion model point alternative mechanism cohesin-dependent organisation. We propose cohesin, akin establishment at replication forks, forms DNA-DNA capture places transcription, thus unifying cohesin’s roles chromosome segregation genome
Язык: Английский
Процитировано
7
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown
Опубликована: Июнь 1, 2024
ABSTRACT The best-studied mechanism of eukaryotic RNA polymerase II (RNAPII) transcriptional termination involves polyadenylation site-directed cleavage the nascent RNA. RNAPII-associated product is then degraded by XRN2, dislodging RNAPII from DNA template. In contrast, prokaryotic RNAP and RNAPIII often terminate directly at T-tracts in coding strand. Here, we demonstrate a similar omnipresent capability for mammalian RNAPII. XRN2- T-tract-dependent are independent - latter usually acting when XRN2 cannot be engaged. We show that snRNA transcription, previously thought to require Integrator complex. Importantly, find genome-wide promoter-proximal regions, but not within protein-coding gene bodies. XRN2-dependent dominates downstream genes, T-tract process sometimes employed. Overall, global DNA-directed attrition suggesting RNAPs retain potential over T-rich sequences throughout evolution.
Язык: Английский
Процитировано
2
Nature Communications, Год журнала: 2024, Номер 15(1)
Опубликована: Сен. 8, 2024
Язык: Английский
Процитировано
2
Genes & Development, Год журнала: 2024, Номер 38(21-24), С. 998 - 1019
Опубликована: Ноя. 1, 2024
The best-studied mechanism of eukaryotic RNA polymerase II (RNAPII) transcriptional termination involves polyadenylation site-directed cleavage the nascent RNA. RNAPII-associated product is then degraded by XRN2, dislodging RNAPII from DNA template. In contrast, prokaryotic RNAP and RNAPIII often terminate directly at T-tracts in coding strand. Here, we demonstrate a similar omnipresent capability for mammalian RNAPII. Importantly, this does not require upstream cleavage. Accordingly, T-tract-dependent can take place when XRN2 cannot be engaged. We show that snRNA transcription independently Integrator complex. found genome-wide promoter-proximal regions but within protein-coding gene bodies. XRN2-dependent dominates downstream genes, T-tract process sometimes used. Overall, global DNA-directed attrition transcription, suggesting RNAPs retain potential to over T-rich sequences throughout evolution.
Язык: Английский
Процитировано
2
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown
Опубликована: Окт. 16, 2023
SUMMARY The timely termination of RNA polymerase II (Pol II) transcription is critical for recycling and preventing interference with the expression neighbouring genes. Termination Pol involves exoribonucleolytic decay nascent by 5’-3’ exonuclease Xrn2. Xrn2 attacks 5’-PO 4 -end executes degradation generated endonucleolytic cleavage at poly(A) site which eventually leads to release from DNA. However, molecular details when how during this process interacts elongation complex mediate its dissociation DNA not understood. Here, we demonstrate that Spt5, a conserved factor controls processivity pausing. Importantly, activity stimulated Spt5 in vitro Spt5-depleted cells show defective termination. Our results support model where first forms stable elongating acquire full degrading RNA. also promotes premature attenuating non-coding transcripts. By contrast, depletion retention promoters protein-coding IIs transcribe into gene body absence exhibit severely reduced rates pre-mRNA processing. We propose plays major role production functional mRNA directly stimulating enzymes entry complexes configured processing elongation.
Язык: Английский
Процитировано
5
Science Advances, Год журнала: 2024, Номер 10(34)
Опубликована: Авг. 23, 2024
RNA polymerase IV (Pol IV) forms a complex with RNA-directed 2 (RDR2) to produce double-stranded (dsRNA) precursors essential for plant gene silencing. In the “backtracking-triggered channeling” model, Pol backtracks and delivers its transcript’s 3′ terminus RDR2, which synthesizes dsRNA. However, mechanisms underlying backtracking protection from cleavage are unclear. Here, we determined cryo–electron microscopy structures of elongation complexes at four states nucleotide addition cycle (NAC): posttranslocation, guanosine triphosphate–bound, pretranslocation, backtracked states. The reveal that maintains an open DNA cleft kinked bridge helix in all NAC states, loosely interacts nucleoside triphosphate substrate, barely contacts proximal nucleotides. Biochemical data indicate is inefficient forward translocation cleavage. These findings suggest transcription prone incapable hydrolysis, ensuring efficient dsRNA production by IV–RDR2.
Язык: Английский
Процитировано
1
Journal of Molecular Biology, Год журнала: 2024, Номер unknown, С. 168814 - 168814
Опубликована: Окт. 1, 2024
The accurate and efficient biogenesis of RNA by cellular polymerase (RNAP) requires accessory factors that regulate the initiation, elongation, termination transcription. Of many discovered to date, elongation regulator NusG-Spt5 is only universally conserved transcription factor. With orthologs paralogs found in all three domains life, this ubiquity underscores their ancient essential regulatory functions. proteins evolved maintain a similar binding interface RNAP through contacts NusG N-terminal domain (NGN) bridge main DNA-binding cleft. We propose varying strength these contacts, modulated tethering interactions, either decrease transcriptional pausing smoothing rugged thermodynamic landscape transcript or enhance pausing, depending on which conformation stabilized NGN contacts. contains one (in bacteria archaea) more eukaryotes) C-terminal use KOW fold contact diverse targets, tether NGN, control biogenesis. Recent work highlights functions different organisms. Some contain multiple specialized subsets operons via sequence-specific targeting, controlling production antibiotics, toxins, capsule proteins. Despite common origin, can differ target selection, interacting partners, effects synthesis. describe current understanding structure, interactions with other regulators, including significant recent progress from genome-wide analyses, single-molecule visualization, cryo-EM. findings highlight remarkable diversity function among structurally
Язык: Английский
Процитировано
1
Structure, Год журнала: 2024, Номер unknown
Опубликована: Дек. 1, 2024
Язык: Английский
Процитировано
1
Yeast, Год журнала: 2023, Номер 41(4), С. 186 - 191
Опубликована: Дек. 2, 2023
Polyadenylation occurs at numerous sites within 3'-untranslated regions (3'-UTRs) but rarely coding regions. How does Pol II travel through long without generating poly(A) sites, yet then permits promiscuous polyadenylation once it reaches the 3'-UTR? The cleavage/polyadenylation (CpA) machinery preferentially associates with 3'-UTRs, is unknown how its recruitment restricted to 3'-UTRs during elongation. Unlike regions, have AT-rich stretches of DNA that may be important for restricting 3'-UTRs. Recognition 3'-UTR could occur (AT-rich), RNA (AU-rich), or RNA:DNA hybrid (rU:dA- and/or rA:dT-rich) level. Based on nucleic acid critical recognition, there are three classes models, not mutually exclusive, CpA selectively recruited thereby where occurs: (1) RNA-based models suggest complex directly (or indirectly one more intermediary proteins) binds AU-rich exposed after passes these (2) DNA-based sequence affects nucleosome depletion elongating machinery, resulting in dissociation some elongation factors and subsequent machinery. (3) preferential destabilization rU:dA- rA:dT-rich duplexes bridging nucleotide addition exit association Experiments provide evidence suggested.
Язык: Английский
Процитировано
2