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.

Pause, Arnim et al. (2015) International Worm Meeting "Loss of Folliculin confers osmotic stress resistance via AMPK-dependent remodeling of carbohydrate stores in C. elegans."

Pause, Arnim, Possik, Elite, Manteghi, Sanaz, Flamand, Mathieu, Coull, Barry, van Stensel, Maurice, Hall, David, Vijayaraghavan, Tarika, Ajisebutu, Andrew

[International Worm Meeting, 2015]

Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular life. When exposed to hyperosmotic stress, cells and organisms utilize conserved strategies to prevent water loss and maintain cellular integrity and viability. Here we identify a novel AMPK-dependent pathway of resistance to hypertonic stress mediated by the AMPK regulator flcn-1 in C. elegans. FLCN-1 is the worm homologue of the tumor suppressor Folliculin (FLCN), responsible for the Birt-Hogg-Dube hereditary cancer disorder. We show that loss of flcn-1 increases glycogen stores in an AMPK dependent manner and that the glycogen reserves are rapidly degraded upon hypertonic stress, leading to a remarkable accumulation of the organic osmolyte glycerol, promoting resistance to hyperosmotic stress. Importantly, loss of AMPK, glycogen synthase or glycogen phospharylase, which are critical enzymes in glycogen metabolism, strongly suppressed the increased osmotic stress resistance in flcn mutant animals. We further demonstrate that glycerol 3-phosphate dehydrogenase-1 is strongly induced in flcn-1 animals upon hyperosmotic stress and that simultaneous loss of gpdh-1 and gpdh-2 abolished the flcn-1/AMPK-mediated osmotic stress phenotype. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in kidneys and renal tumors from a BHD patient. Since BHD is a renal hyperproliferation disorder, a mechanism of osmotic stress resistance in kidney hyperosmotic environments might explain tumorigenesis in BHD patients. Overall, our data indicate that FLCN is an evolutionary conserved regulator of glycogen metabolism, that might be acting as a tumor suppressor via AMPK-dependent accumulation of glycogen and organic osmolytes, resulting in an advantageous increase in proliferation and survival in hyperosmotic environments. .

Maxwell, Colin S. et al. (2013) International Worm Meeting "Poising and pausing of Pol II during starvation."

Meyer, Barbara J., Antoshechkin, Igor, Baugh, L. Ryan, Kurhanewicz, Nicole, Lewarch, Caitlin L., Lis, John T., Maxwell, Colin S., Kruesi, William S., Core, Leighton J., Waters, Colin T.

[International Worm Meeting, 2013]

In order to respond appropriately to changing environments, organisms must quickly alter the genes they express. Arrested L1 larvae rapidly alter gene expression in response to feeding, providing an attractive model to study the mechanisms of rapid gene induction. We previously showed that RNA Polymerase II (Pol II) is regulated at a post-recruitment step during L1 arrest. This regulation correlates with genes up-regulated by feeding, suggesting that it promotes rapid gene induction. We hypothesized that this regulation was mechanistically related to Pol II pausing, which has been proposed to allow the rapid induction of genes. To address this, we located elongation complexes genome-wide during starvation by sequencing short nascent RNAs as well as by using global nuclear run-on sequencing. We show here that Pol II is regulated during early elongation (pausing). Analysis of a TFIIS mutant reveals mechanistic similarities to pausing in other systems. However, Pol II pausing is associated with active stress-response genes that are actually down-regulated upon feeding. In addition to pausing, we show that 'poised' Pol II accumulates without initiating upstream of repressed growth genes that are up-regulated upon feeding. Poised Pol II and paused Pol II are associated with distinct core promoter architectures, suggesting alternative pathways for pre-initiation complex formation. Both growth and stress genes are regulated post-recruitment during starvation, but during initiation and elongation, respectively. Our work sheds light on the mechanisms organisms use to cope with changing environments.

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.

Blumberg A et al. (2019) Genome Res "Corrigendum: Initiation of mtDNA transcription is followed by pausing, and diverges across human cell types and during evolution."

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

[Genome Res, 2019]

Katz, Menachem et al. (2011) International Worm Meeting "C. elegans locomotory pattern, pausing frequency and speed is regulated by the CEP sheath glia."

Corson, Francis, Shaham, Shai, Katz, Menachem

[International Worm Meeting, 2011]

Neuronal circuits are tightly regulated to achieve proper animal behavior. Recent studies suggest that glial cells can influence neuronal functions and synaptic activities, and may, thus, contribute to behavior. However, the precise contributions of synaptic glia to animal behavior are poorly understood. C. elegans glia share morphological, functional, and genetic features with their vertebrate counterparts. However, in contrast to vertebrate glia, C. elegans glia are not essential for neuronal survival, offering a unique arena for exploring the involvement of glia in neuronal functions in a live animal. The CEP sheath glia (CEPsh) are bipolar cells that ensheath the dendrites of CEP neurons, and envelope the nerve ring. Reminiscent of mammalian astrocytes, these cells extend processes that abut specific synapses within the nerve ring. We showed that ablation of the CEPsh glia during the first larval stage results in abnormal locomotory behavior. CEPsh-ablated animals display reduced locomotion speed and extended locomotory pausing, as well as exaggerated small-angle turns and frequent reversals that limit their dispersal. To understand how these behaviors are regulated by CEPsh glia, we focused on the ALA-AVE synapse ensheathed by these glia (White et al., 1986). In line with the previously described role of ALA in behavioral quiescence and locomotory pausing (Van Buskirk and Sternberg, 2008), we found that inactivation of ALA reduces the pausing frequencies of glia-ablated animals. In addition, the dispersal of these animals and their speed of locomotion are improved. Inactivation of AVE in wild-type animals, induces extended pausing periods comparable to those seen in glia ablated animals, and animal speed is reduced. Our results suggest that AVE is a key regulator of speed and pausing frequency in the C. elegans nervous system, and that ALA functions to inhibit the activity of AVE. Moreover, our results indicate that the CEPsh glia provide important negative regulation on the activity of this tripartite synapse. To understand the molecular basis of CEPsh glia function we have begun to identify genes expressed in these cells using an RNA-tagging method, with the aim of examining the roles of enriched genes in C. elegans locomotory behavior.

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.