Stein KC et al. (2022) Nature "Ageing exacerbates ribosome pausing to disrupt cotranslational proteostasis."

Frydman J, Stein KC, Morales-Polanco F, van der Lienden J, Rainbolt TK

[Nature, 2022]

Ageing is accompanied by a decline in cellular proteostasis, which underlies many age-related protein misfolding diseases^1,2. Yet, how ageing impairs proteostasis remains unclear. As nascent polypeptides represent a substantial burden on the proteostasis network³, we hypothesized that altered translational efficiency during ageing could help to drive the collapse of proteostasis. Here we show that ageing alters the kinetics of translation elongation in both Caenorhabditis elegans and Saccharomyces cerevisiae. Ribosome pausing was exacerbated at specific positions in aged yeast and worms, including polybasic stretches, leading to increased ribosome collisions known to trigger ribosome-associated quality control (RQC)^4-6. Notably, aged yeast cells exhibited impaired clearance and increased aggregation of RQC substrates, indicating that ageing overwhelms this pathway. Indeed, long-lived yeast mutants reduced age-dependent ribosome pausing, and extended lifespan correlated with greater flux through the RQC pathway. Further linking altered translation to proteostasis collapse, we found that nascent polypeptides exhibiting age-dependent ribosome pausing in C. elegans were strongly enriched among age-dependent protein aggregates. Notably, ageing increased the pausing and aggregation of many components of proteostasis, which could initiate a cycle of proteostasis collapse. We propose that increased ribosome pausing, leading to RQC overload and nascent polypeptide aggregation, critically contributes to proteostasis impairment and systemic decline during ageing.

Maxwell CS et al. (2014) Cell Rep "Pol II docking and pausing at growth and stress genes in C. elegans."

Maxwell CS, Meyer BJ, Kruesi WS, Lewarch CL, Kurhanewicz N, Baugh LR, Lis JT, Core LJ, Waters CT, Antoshechkin I

[Cell Rep, 2014]

Fluctuations in nutrient availability profoundly impact gene expression. Previous work revealed postrecruitment regulation of RNA polymerase II (Pol II) during starvation and recovery in Caenorhabditis elegans, suggesting that promoter-proximal pausing promotes rapid response to feeding. To test this hypothesis, we measured Pol II elongation genome wide by two complementary approaches and analyzed elongation in conjunction with Pol II binding and expression. We confirmed bona fide pausing during starvation and also discovered Pol II docking. Pausing occurs at active stress-response genes that become downregulated in response to feeding. In contrast, "docked" Pol II accumulates without initiating upstream of inactive growth genes that become rapidly upregulated upon feeding. Beyond differences in function and expression, these two sets of genes have different core promoter motifs, suggesting alternative transcriptional machinery. Our work suggests that growth and stress genes are both regulated postrecruitment during starvation but at initiation and elongation, respectively, coordinating gene expression with nutrient availability.

Blumberg A et al. (2017) Genome Res "Initiation of mtDNA transcription is followed by pausing, and diverge across human cell types and during evolution."

Danko CG, Rice EJ, Mishmar D, Kundaje A, Blumberg A

[Genome Res, 2017]

Mitochondrial DNA (mtDNA) genes are long known to be co-transcribed in polycistrones, yet it remains impossible to study nascent mtDNA transcripts quantitatively in vivo using existing tools. To this end we used deep sequencing (GRO-seq and PRO-seq) and analyzed nascent mtDNA-encoded RNA transcripts in diverse human cell lines and metazoan organisms. Surprisingly, accurate detection of human mtDNA transcription initiation sites (TIS) in the heavy and light strands revealed a novel conserved transcription pausing site near the light strand TIS. This pausing site correlated with the presence of a bacterial pausing sequence motif, reduced SNP density, and with a DNase footprinting signal in all tested cells. Its location within conserved sequence block 3 (CSBIII), just upstream of the known transcription-replication transition point suggests involvement in such transition. Analysis of non-human organisms enabled de novo mtDNA sequence assembly, as well as detection of previously unknown mtDNA TIS, pausing, and transcription termination sites with unprecedented accuracy. Whereas mammals (Pan troglodytes, Macaca mulatta, Rattus norvegicus, and Mus musculus) showed a human-like mtDNA transcription pattern, the invertebrate pattern (Drosophila melanogaster and Caenorhabditis elegans) profoundly diverged. Our approach paves the path towards in vivo, quantitative, reference sequence-free analysis of mtDNA transcription in all eukaryotes.

Dollinger R et al. (2022) FASEB J "Spt5-nucleic acid interactions are directly involved in promoter proximal pausing of RNA polymerase II."

Dollinger R, Gilmour DS

[FASEB J, 2022]

Promoter proximal pausing of RNA polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires DSIF and NELF. DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. We use Drosophila melanogasteras a model system to address this question. We studied DSIF-nucleic acid contacts made by the Spt5 NGN and KOW1 domains, which form a part of the upstream DNA exit tunnel, and the Spt5 KOW4 domain, which, along with the KOW5 domain, forms a clamp around the nascent RNA. Biochemical and structural studies have implicated these domains in pausing. We hypothesize that electrostatic interactions between Spt5 and the nucleic acid scaffold contribute to pausing by inhibiting the translocation of Pol II. We disrupted these electrostatic interactions by reversing the charge of key basic residues in Spt5 and used an electrophoretic mobility shift assay to measure Pol II binding and NELF recruitment. We then tested the pausing activity of the DSIF mutants in Drosophilanuclear extract depleted of wild-type DSIF. Reversing the charge of six basic residues in the KOW1 domain resulted in weaker binding to Pol II. Though this mutant was still able to recruit NELF, it showed greatly reduced pausing activity. Reversing the charge of four basic residues in the KOW4 domain only modestly reduced binding to Pol II, had no impact on NELF recruitment, and significantly impaired DSIF's pausing function. Thus, nucleic acid contacts made by the KOW1 domain of Spt5 promote pausing by mediating Pol II-DSIF interactions (and therefore NELF recruitment) while the KOW4 domain interactions with the nascent transcript contribute to pausing directly. We also reversed the charge of two basic residues in the NGN domain and found that these changes had no effect on DSIF binding and NELF recruitment but did result in a modest decrease in pausing activity. One of these basic residues is an arginine located in an alpha helix sequence that is conserved in eukaryotes with NELF and promoter proximal pausing but is absent in eukaryotes lacking NELF. To test whether this alpha helix is required for promoter proximal pausing, we replaced the Drosophila helix sequence with unstructured loop sequences from Komagataella pastoris, Saccharomyces cerevisiae, and Caenorhabditis elegans, none of which have NELF. These mutants were able to bind Pol II and recruit NELF but exhibited significantly reduced pausing activity, suggesting that a short alpha helix in the NGN domain is critical for stabilizing the promoter proximal pause. This is likely achieved by optimally positioning a conserved arginine to interact with the DNA scaffold.

Beltran T et al. (2020) EMBO J "Integrator is recruited to promoter-proximally paused RNA Pol II to generate Caenorhabditis elegans piRNA precursors."

Sarkies P, Beltran T, Pahita E, Ghosh S, Lenhard B

[EMBO J, 2020]

Piwi-interacting RNAs (piRNAs) play key roles in germline development and genome defence in metazoans. In C.elegans, piRNAs are transcribed from >15,000 discrete genomic loci by RNA polymerase II (Pol II), resulting in 28 nt short-capped piRNA precursors. Here, we investigate transcription termination at piRNA loci. We show that the Integrator complex, which terminates snRNA transcription, is recruited to piRNA loci. Moreover, we demonstrate that the catalytic activity of Integrator cleaves nascent capped piRNA precursors associated with promoter-proximal Pol II, resulting in termination of transcription. Loss of Integrator activity, however, does not result in transcriptional readthrough at the majority of piRNA loci. Taken together, our results draw new parallels between snRNA and piRNA biogenesis in nematodes and provide evidence of a role for the Integrator complex as a terminator of promoter-proximal RNA polymerase II during piRNA biogenesis.

Beltran T et al. (2019) Dev Cell "Comparative Epigenomics Reveals that RNA Polymerase II Pausing and Chromatin Domain Organization Control Nematode piRNA Biogenesis."

Beltran T, Martinez-Perez E, Stevens L, Fradin H, Piano F, Birkle TY, Schwartz HT, Sternberg PW, Barroso C, Sarkies P, Ahringer J, Gunsalus K, Blaxter M, Cerrato C, Sharma G

[Dev Cell, 2019]

Piwi-interacting RNAs (piRNAs) are important for genome regulation across metazoans, but their biogenesis evolves rapidly. In Caenorhabditis elegans, piRNA loci are clustered within two 3-Mb regions on chromosome IV. Each piRNA locus possesses an upstream motif that recruits RNA polymerase II to produce an 28 nt primary transcript. We used comparative epigenomics across nematodes to gain insight into the origin, evolution, and mechanism of nematode piRNA biogenesis. We show that the piRNA upstream motif is derived from core promoter elements controlling snRNA transcription. We describe two alternative modes of piRNA organization in nematodes: in C.elegans and closely related nematodes, piRNAs are clustered within repressive H3K27me3 chromatin, while in other species, typified by Pristionchus pacificus, piRNAs are found within introns of active genes. Additionally, we discover that piRNA production depends on sequence signals associated with RNA polymerase II pausing. We show that pausing signals synergize with chromatin to control piRNA transcription.

Macaisne N et al. (2023) Elife "Synergistic stabilization of microtubules by BUB-1, HCP-1 and CLS-2 controls microtubule pausing and meiotic spindle assembly."

Bellutti L, Geoffroy H, Gervais A, Pitayu-Nugroho L, Canman JC, Maton G, Dumont J, Laband K, Macaisne N, Lacroix B, Ganeswaran T, Edwards F

[Elife, 2023]

During cell division, chromosome segregation is orchestrated by a microtubule-based spindle. Interaction between spindle microtubules and kinetochores is central to the bi-orientation of chromosomes. Initially dynamic to allow spindle assembly and kinetochore attachments, which is essential for chromosome alignment, microtubules are eventually stabilized for efficient segregation of sister chromatids and homologous chromosomes during mitosis and meiosis I respectively. Therefore, the precise control of microtubule dynamics is of utmost importance during mitosis and meiosis. Here, we study the assembly and role of a kinetochore module, comprised of the kinase BUB-1, the two redundant CENP-F orthologs HCP-1/2, and the CLASP family member CLS-2 (hereafter termed the BHC module), in the control of microtubule dynamics in Caenorhabditis elegans oocytes. Using a combination of in vivo structure-function analyses of BHC components and in vitro microtubule-based assays, we show that BHC components stabilize microtubules, which is essential for meiotic spindle formation and accurate chromosome segregation. Overall, our results show that BUB-1 and HCP-1/2 do not only act as targeting components for CLS-2 at kinetochores, but also synergistically control kinetochore-microtubule dynamics by promoting microtubule pause. Together, our results suggest that BUB-1 and HCP-1/2 actively participate in the control of kinetochore-microtubule dynamics in the context of an intact BHC module to promote spindle assembly and accurate chromosome segregation in meiosis.

Stubbe FX et al. (2025) Nat Commun "SSUP-72/PINN-1 coordinates RNA-polymerase II 3' pausing and developmental gene expression in C. elegans."

Stubbe FX, Ponsard P, Hermand D, Steiner FA

[Nat Commun, 2025]

During exit from Caenorhabditis elegans (C. elegans) L1 developmental arrest, a network of growth- and developmental genes is activated, many of which are organized into operons where transcriptional termination is uncoupled from mRNA 3'-end processing. CDK-12-mediated Pol II CTD S2 phosphorylation enhances SL2 trans-splicing at downstream operonic genes, preventing premature termination and ensuring proper gene expression for developmental progression. Using a genetic screen, we identified the SSUP-72/PINN-1 module as a suppressor of defects induced by CDK-12 inhibition. Loss of SSUP-72/PINN-1 bypasses the requirement for CDK-12 in post-embryonic development. Genome-wide analyses reveal that SSUP-72, a CTD S5P phosphatase, affects Pol II 3' pausing and regulates intra-operon termination. Our findings establish SSUP-72/PINN-1 as a key regulator of Pol II dynamics, coordinating operonic gene expression and growth during C. elegans post-embryonic development.

Gramlich MW et al. (2021) Sci Rep "Distinguishing synaptic vesicle precursor navigation of microtubule ends with a single rate constant model."

Balseiro-Gomez S, Yogev S, Parkes M, Gramlich MW, Tabei SMA

[Sci Rep, 2021]

Axonal motor driven cargo utilizes the microtubule cytoskeleton in order to direct cargo, such as synaptic vesicle precursors (SVP), to where they are needed. This transport requires vesicles to travel up to microns in distance. It has recently been observed that finite microtubule lengths can act as roadblocks inhibiting SVP and increasing the time required for transport. SVPs reach the end of a microtubule and pause until they can navigate to a neighboring microtubule in order to continue transport. The mechanism(s) by which axonal SVPs navigate the end of a microtubule in order to continue mobility is unknown. In this manuscript we model experimentally observed vesicle pausing at microtubule ends in C. elegans. We show that a single rate-constant model reproduces the time SVPs pause at MT-ends. This model is based on the time an SVP must detach from its current microtubule and re-attach to a neighboring microtubule. We show that vesicle pause times are different for anterograde and retrograde motion, suggesting that vesicles utilize different proteins at plus and minus end sites. Last, we show that vesicles do not likely utilize a tug-of-war like mechanism and reverse direction in order to navigate microtubule ends.

Yogev S et al. (2016) Neuron "Microtubule Organization Determines Axonal Transport Dynamics."

Horowitz M, Yogev S, Cooper R, Fetter R, Shen K

[Neuron, 2016]

Axonal microtubule (MT) arrays are the major cytoskeleton substrate for cargo transport. How MTorganization, i.e., polymer length, number, and minus-end spacing, is regulated and how it impinges on axonal transport are unclear. We describe a method for analyzing neuronal MT organization usinglight microscopy. This method circumvents the needfor electron microscopy reconstructions and is compatible with live imaging of cargo transport and MT dynamics. Examination of a C.elegans motor neuron revealed how age, MT-associated proteins, and signaling pathways control MT length, minus-end spacing, and coverage. In turn, MT organization determines axonal transport progression: cargoes pause at polymer termini, suggesting that switching MT tracks is rate limiting for efficient transport. Cargo run length is set by MT length, and higher MT coverage correlates with shorter pauses. These results uncover the principles and mechanisms of neuronal MT organization and its regulation of axonal cargo transport.