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1. The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

   https://doi.org/10.1073/pnas.1915312116  

   Song, Jiarui ; Logeswaran, Dhenugen ; Castillo-González, Claudia ; Li, Yang ; Bose, Sreyashree ; Aklilu, Behailu Birhanu ; Ma, Zeyang ; Polkhovskiy, Alexander ; Chen, Julian J.-L. ; Shippen, Dorothy E. ( December 2019 , Proceedings of the National Academy of Sciences)

   null (Ed.)

   Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana , called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3′ long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates. 

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2. Plant telomere biology: The green solution to the end-replication problem

   https://doi.org/10.1093/plcell/koac122  

   Shakirov, Eugene V. ; Chen, Julian J. -L ; Shippen, Dorothy E. ( May 2022 , The Plant Cell)

   Telomere maintenance is a fundamental cellular process conserved across all eukaryotic lineages. Although plants and animals diverged over 1.5 billion years ago, lessons learned from plants continue to push the boundaries of science, revealing detailed molecular mechanisms in telomere biology with broad implications for human health, aging biology, and stress responses. Recent studies of plant telomeres have unveiled unexpected divergence in telomere sequence and architecture, and the proteins that engage telomeric DNA and telomerase. The discovery of telomerase RNA components in the plant kingdom and some algae groups revealed new insight into the divergent evolution and the universal core of telomerase across major eukaryotic kingdoms. In addition, resources cataloging the abundant natural variation in Arabidopsis thaliana, maize (Zea mays), and other plants are providing unparalleled opportunities to understand the genetic networks that govern telomere length polymorphism and, as a result, are uncovering unanticipated crosstalk between telomeres, environmental factors, organismal fitness, and plant physiology. Here we recap current advances in plant telomere biology and put this field in perspective relative to telomere and telomerase research in other eukaryotic lineages.

    

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3. Biogenesis of telomerase RNA from a protein-coding mRNA precursor

   https://doi.org/10.1073/pnas.2204636119  

   Logeswaran, Dhenugen ; Li, Yang ; Akhter, Khadiza ; Podlevsky, Joshua D. ; Olson, Tamara L. ; Forsberg, Katherine ; Chen, Julian J.-L. ( October 2022 , Proceedings of the National Academy of Sciences)

   Telomerase is a eukaryotic ribonucleoprotein (RNP) enzyme that adds DNA repeats onto chromosome ends to maintain genomic stability and confer cellular immortality in cancer and stem cells. The telomerase RNA (TER) component is essential for telomerase catalytic activity and provides the template for telomeric DNA synthesis. The biogenesis of TERs is extremely divergent across eukaryotic kingdoms, employing distinct types of transcription machinery and processing pathways. In ciliates and plants, TERs are transcribed by RNA polymerase III (Pol III), while animal and ascomycete fungal TERs are transcribed by RNA Pol II and share biogenesis pathways with small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA), respectively. Here, we report an unprecedented messenger RNA (mRNA)-derived biogenesis pathway for the 1,291 nucleotide TER from the basidiomycete fungus Ustilago maydis . The U. maydis TER ( Um TER) contains a 5′-monophosphate, distinct from the 5′ 2,2,7-trimethylguanosine (TMG) cap common to animal and ascomycete fungal TERs. The mature Um TER is processed from the 3′-untranslated region (3′-UTR) of a larger RNA precursor that possesses characteristics of mRNA including a 5′ 7-methyl-guanosine (m 7 G) cap, alternative splicing of introns, and a poly(A) tail. Moreover, this mRNA transcript encodes a protein called Early meiotic induction protein 1 (Emi1) that is conserved across dikaryotic fungi. A recombinant Um TER precursor expressed from an mRNA promoter is processed correctly to yield mature Um TER, confirming an mRNA-processing pathway for producing TER. Our findings expand the plethora of TER biogenesis mechanisms and demonstrate a pathway for producing a functional long noncoding RNA from a protein-coding mRNA precursor. 

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4. Proteome Profile of Endogenous Retrotransposon‐Associated Complexes in Human Embryonic Stem Cells

   https://doi.org/10.1002/pmic.201900169  

   Vuong, Linh M. ; Pan, Songqin ; Donovan, Peter J. ( July 2019 , PROTEOMICS)

   Long Interspersed Element‐1 (LINE‐1 or L1) are transposable elements similar to retroviruses that have existed in the genome of primates for millions of years. They encode two Open Reading Frame (ORF) proteins (ORF1p and ORF2p) that bind L1 RNA to form a ribonucleoprotein (RNP) complex and are required for L1 integration into the host genome. Humans have evolved with L1 and found ways to limit L1 activity. To identify partners of the L1 RNP, previous studies used ectopic expression of L1 ORF1/2p or RNA in various cancer cells, which express low levels of the ORF proteins. Whether naturally occurring L1 RNP interacts with the same proteins in non‐cancer cells is unknown. Here, the aim is to examine the natural assembly of endogenous L1 RNPs in normal human cells. L1 elements are expressed in human embryonic stem cells (hESCs), derived from pre‐implantation embryos. Therefore, these cells are used to immunoprecipitate ORF1p followed by MS to identify proteins that associate with the naturally‐occurring L1 ORF1p. Some of the same proteins as well as unique proteins are found interacting with the endogenous L1 ORF1p complexes. The analysis of ORF1p‐associated proteins in hESCs can help address important questions in both retrotransposon biology and the biology of hESCs.

    

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5. A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities

   https://doi.org/10.3389/fpls.2021.707378  

   Diwan, Danish ; Liu, Xiaoyu ; Andrews, Caroline F. ; Pajerowska-Mukhtar, Karolina M. ( July 2021 , Frontiers in Plant Science)

   The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 ( basic leucine zipper 60 ) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro , the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta . Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues. 

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