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1. A degenerate telomerase RNA directs telomeric DNA synthesis in lepidopteran insects

   https://doi.org/10.1073/pnas.2424443122  

   Chou, Yu-Shu; Logeswaran, Dhenugen; Chow, Chi-Nga; Dunn, Phoebe; Podlevsky, Joshua D; Liu, Tianxiang; Akhter, Khadiza; Chen, Julian J-L (March 2025, Proceedings of the National Academy of Sciences)

   Feigon, Juli (Ed.)

   Telomerase elongates telomeres to maintain chromosome stability in most eukaryotes. Despite extensive studies across eukaryotic kingdoms, the telomerase holoenzyme in arthropods remains poorly understood. In this study, we purify the telomerase ribonucleoprotein complex from the lepidopteran insectSpodoptera frugiperda(fall armyworm) and identify a copurified 135-nucleotide telomerase RNA (TR) component. This miniatureS. frugiperdaTR (sfTR), the smallest TR known to date, retains a universal pseudoknot structure and a structurally defined template. Despite its small size, sfTR assembles with the recombinantS. frugiperdatelomerase reverse transcriptase (sfTERT) protein in vivo to reconstitute telomerase activity for the synthesis of insect telomeric DNA repeats (TTAGG)n. The sfTR gene, like other animal TR genes, features an snRNA-type RNA polymerase II promoter. Uniquely, the sfTR transcript harbors a 5′-7-methylguanosine (M⁷G) cap, as opposed to the more typical snRNA-type 2,2,7-trimethylguanosine (TMG) cap. The difference in 5′-cap is likely because sfTR lacks the H/ACA snoRNA biogenesis domain necessary for cap hypermethylation. Moreover, sfTR also lacks the CR4/5 regulatory domain that is indispensable in vertebrate TRs for telomerase activity. This degenerate sfTR complements an enigmatic sfTERT that is missing certain telomerase-specific elements yet catalytically active in the absence of sfTR. Thus, insects have evolved a simplified telomerase, consisting of a small noncoding RNA that retains only minimal attributes essential for telomerase function. The simplified insect telomerase demonstrates a plausible evolutionary pathway for the emergence of telomerase ribonucleoprotein complex, arising from an ancient reverse transcriptase associated with a simple templating RNA component in early eukaryotes. 

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2. Monophyletic Origin and Divergent Evolution of Animal Telomerase RNA

   https://doi.org/10.1093/molbev/msaa203  

   Logeswaran, Dhenugen; Li, Yang; Podlevsky, Joshua D; Chen, Julian J (August 2020, Molecular Biology and Evolution)

   Wittkopp, Patricia (Ed.)

   Abstract Telomerase RNA (TR) is a noncoding RNA essential for the function of telomerase ribonucleoprotein. TRs from vertebrates, fungi, ciliates, and plants exhibit extreme diversity in size, sequence, secondary structure, and biogenesis pathway. However, the evolutionary pathways leading to such unusual diversity among eukaryotic kingdoms remain elusive. Within the metazoan kingdom, the study of TR has been limited to vertebrates and echinoderms. To understand the origin and evolution of TR across the animal kingdom, we employed a phylogeny-guided, structure-based bioinformatics approach to identify 82 novel TRs from eight previously unexplored metazoan phyla, including the basal-branching sponges. Synthetic TRs from two representative species, a hemichordate and a mollusk, reconstitute active telomerase in vitro with their corresponding telomerase reverse transcriptase components, confirming that they are authentic TRs. Comparative analysis shows that three functional domains, template-pseudoknot (T-PK), CR4/5, and box H/ACA, are conserved between vertebrate and the basal metazoan lineages, indicating a monophyletic origin of the animal TRs with a snoRNA-related biogenesis mechanism. Nonetheless, TRs along separate animal lineages evolved with divergent structural elements in the T-PK and CR4/5 domains. For example, TRs from echinoderms and protostomes lack the canonical CR4/5 and have independently evolved functionally equivalent domains with different secondary structures. In the T-PK domain, a P1.1 stem common in most metazoan clades defines the template boundary, which is replaced by a P1-defined boundary in vertebrates. This study provides unprecedented insight into the divergent evolution of detailed TR secondary structures across broad metazoan lineages, revealing ancestral and later-diversified elements. 

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3. Mutations in yeast Pcf11, a conserved protein essential for mRNA 3′ end processing and transcription termination, elicit the Environmental Stress Response

   https://doi.org/10.1093/genetics/iyad199  

   Graber, Joel H.; Hoskinson, Derick; Liu, Huiyun; Kaczmarek Michaels, Katarzyna; Benson, Peter S.; Maki, Nathaniel J.; Wilson, Christian L.; McGrath, Caleb; Puleo, Franco; Pearson, Erika; et al (November 2023, GENETICS)

   Abstract The Pcf11 protein is an essential subunit of the large complex that cleaves and polyadenylates eukaryotic mRNA precursor. It has also been functionally linked to gene-looping, termination of RNA Polymerase II (Pol II) transcripts, and mRNA export. We have examined a poorly characterized but conserved domain (amino acids 142–225) of the Saccharomyces cerevisiae  Pcf11 and found that while it is not needed for mRNA 3′ end processing or termination downstream of the poly(A) sites of protein-coding genes, its presence improves the interaction with Pol II and the use of transcription terminators near gene promoters. Analysis of genome-wide Pol II occupancy in cells with Pcf11 missing this region, as well as Pcf11 mutated in the Pol II CTD Interacting Domain, indicates that systematic changes in mRNA expression are mediated primarily at the level of transcription. Global expression analysis also shows that a general stress response, involving both activation and suppression of specific gene sets known to be regulated in response to a wide variety of stresses, is induced in the two pcf11 mutants, even though cells are grown in optimal conditions. The mutants also cause an unbalanced expression of cell wall-related genes that does not activate the Cell Wall Integrity pathway but is associated with strong caffeine sensitivity. Based on these findings, we propose that Pcf11 can modulate the expression level of specific functional groups of genes in ways that do not involve its well-characterized role in mRNA 3′ end processing. 

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4. Mechanistic differences in eukaryotic initiation factor requirements for eIF4GI-driven cap-independent translation of structured mRNAs

   https://doi.org/10.1016/j.jbc.2024.107866  

   Saha, Baishakhi; Haizel, Solomon A; Goss, Dixie J (November 2024, Journal of Biological Chemistry)

   Protein translation is globally downregulated under stress conditions. Many proteins that are synthesized under stress conditions use a cap-independent translation initiation pathway. A subset of cellular mRNAs that encode for these proteins contain stable secondary structures within their 5′UTR, and initiate cap-independent translation using elements called cap-independent translation enhancers or internal ribosome entry sites within their 5′UTRs. The interaction among initiation factors such as eukaryotic initiation factor 4E (eIF4E), eIF4A, and eIF4GI, especially in regulating the eIF4F complex during noncanonical translation initiation of different 5′UTR mRNAs, is poorly understood. Here, equilibrium-binding assays, CD studies and in vitro translation assays were used to elucidate the recruitment of these initiation factors to the highly structured 5′UTRs of fibroblast-growth factor 9 (FGF-9) and hypoxia inducible factor 1 subunit alpha (HIF-1α) encoding mRNAs. We showed that eIF4A and eIF4E enhanced eIF4GI’s binding affinity to the uncapped 5′UTR of HIF-1α mRNA, inducing conformational changes in the protein/RNA complex. In contrast, these factors have no effect on the binding of eIF4GI to the 5′UTR of FGF-9 mRNA. Recently, Izidoro et al. reported that the interaction of 42nt unstructured RNA to human eIF4F complex is dominated by eIF4E and ATP-bound state of eIF4A. Here, we show that structured 5′UTR mRNA binding mitigates this requirement. Based on these observations, we describe two possible cap-independent translation mechanisms for FGF-9 and HIF-1α encoding mRNAs used by cells to mitigate cellular stress conditions. 

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5. Ustilago maydis telomere protein Pot1 harbors an extra N-terminal OB fold and regulates homology-directed DNA repair factors in a dichotomous and context-dependent manner

   https://doi.org/10.1371/journal.pgen.1010182  

   Zahid, Syed; Aloe, Sarah; Sutherland, Jeanette H.; Holloman, William K.; Lue, Neal F. (May 2022, PLOS Genetics)

   Zhou, Jin-Qiu (Ed.)

   The telomere G-strand binding protein Pot1 plays multifaceted roles in telomere maintenance and protection. We examined the structure and activities of Pot1 in Ustilago maydis , a fungal model that recapitulates key features of mammalian telomere regulation. Compared to the well-characterized primate and fission yeast Pot1 orthologs, Um Pot1 harbors an extra N-terminal OB-fold domain (OB-N), which was recently shown to be present in most metazoans. Um Pot1 binds directly to Rad51 and regulates the latter’s strand exchange activity. Deleting the OB-N domain, which is implicated in Rad51-binding, caused telomere shortening, suggesting that Pot1-Rad51 interaction facilitates telomere maintenance. Depleting Pot1 through transcriptional repression triggered growth arrest as well as rampant recombination, leading to multiple telomere aberrations. In addition, telomere repeat RNAs transcribed from both the G- and C-strand were dramatically up-regulated, and this was accompanied by elevated levels of telomere RNA-DNA hybrids. Telomere abnormalities of pot1 -deficient cells were suppressed, and cell viability was restored by the deletion of genes encoding Rad51 or Brh2 (the BRCA2 ortholog), indicating that homology-directed repair (HDR) proteins are key mediators of telomere aberrations and cellular toxicity. Together, these observations underscore the complex physical and functional interactions between Pot1 and DNA repair factors, leading to context-dependent and dichotomous effects of HDR proteins on telomere maintenance and protection. 

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