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1. Identification of Novel 5′ and 3′ Translation Enhancers in Umbravirus-Like Coat Protein-Deficient RNA Replicons

   https://doi.org/10.1128/jvi.01736-21  

   Liu, Jingyuan ; Simon, Anne E. ( April 2022 , Journal of Virology)

   Dutch, Rebecca Ellis (Ed.)

   ABSTRACT Translation of plant plus-strand RNA viral genomes that lack a 5′ cap frequently requires the use of cap-independent translation enhancers (CITEs) located in or near the 3′ untranslated region (UTR). 3′CITEs are grouped based on secondary structure and ability to interact with different translation initiation factors or ribosomal subunits, which assemble a complex at the 3′ end that is nearly always transferred to the 5′ end via a long-distance kissing-loop interaction between sequences in the 3′CITE and 5′ hairpins. We report here the identification of a novel 3′CITE in coat protein-deficient RNA replicons that are related to umbraviruses. Umbra-like associated RNAs (ulaRNAs), such as citrus yellow vein-associated virus (CYVaV), are a new type of subviral RNA that do not encode movement proteins, coat proteins, or silencing suppressors but can independently replicate using their encoded RNA-dependent RNA polymerase. An extended hairpin structure containing multiple internal loops in the 3′ UTR of CYVaV is strongly conserved in the most closely related ulaRNAs and structurally resembles an I-shaped structure (ISS) 3′CITE. However, unlike ISS, the CYVaV structure binds to eIF4G and no long-distance interaction is discernible between the CYVaV ISS-like structure and sequences at or near the 5′ end. We also report that the ∼30-nucleotide (nt) 5′ terminal hairpin of CYVaV and related ulaRNAs can enhance translation of reporter constructs when associated with either the CYVaV 3′CITE or the 3′CITEs of umbravirus pea enation mosaic virus (PEMV2) and even independent of a 3′CITE. These findings introduce a new type of 3′CITE and provide the first information on translation of ulaRNAs. IMPORTANCE Umbra-like associated RNAs (ulaRNAs) are a recently discovered type of subviral RNA that use their encoded RNA-dependent RNA polymerase for replication but do not encode any coat proteins, movement proteins, or silencing suppressors yet can be found in plants in the absence of any discernible helper virus. We report the first analysis of their translation using class 2 ulaRNA citrus yellow vein-associated virus (CYVaV). CYVaV uses a novel eIF4G-binding I-shaped structure as its 3′ cap-independent translation enhancer (3′CITE), which does not connect with the 5′ end by a long-distance RNA:RNA interaction that is typical of 3′CITEs. ulaRNA 5′ terminal hairpins can also enhance translation in association with cognate 3′CITEs or those of related ulaRNAs and, to a lesser extent, with 3′CITEs of umbraviruses, or even independent of a 3′CITE. These findings introduce a new type of 3′CITE and provide the first information on translation of ulaRNAs. 

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2. Opium Poppy Mosaic Virus Has an Xrn-Resistant, Translated Subgenomic RNA and a BTE 3′ CITE

   https://doi.org/10.1128/JVI.02109-20  

   Ilyas, Muhammad ; Du, Zhiyou ; Simon, Anne E. ( April 2021 , Journal of Virology)

   Dutch, Rebecca Ellis. (Ed.)

   ABSTRACT Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae . OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNA D ). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3′ untranslated region of OPMV contains two 3′ cap-independent translation enhancers (3′ CITEs), a T-shaped structure (TSS) near its 3′ end, and a Barley yellow dwarf virus -like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5′ sequences in vivo , which differs from how umbravirus 3′ CITEs were used in a previous study. Similarly to most 3′ CITEs, the OPMV BTE links to the 5′ end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence. IMPORTANCE Opium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae . We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5′ end just upstream from a previously predicted xrRNA D site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNA D sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo . We also characterized the OPMV BTE structure, a 3′ cap-independent translation enhancer (3′ CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3′ CITEs. 

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3. A forward genetic screen in C. elegans identifies conserved residues of spliceosomal proteins PRP8 and SNRNP200/BRR2 with a role in maintaining 5′ splice site identity

   https://doi.org/10.1093/nar/gkac991  

   Cartwright-Acar, Catiana H. ; Osterhoudt, Kenneth ; Suzuki, Jessie M. N. G. L. ; Gomez, Destiny R. ; Katzman, Sol ; Zahler, Alan M. ( November 2022 , Nucleic Acids Research)

   The spliceosome undergoes extensive rearrangements as it assembles onto precursor messenger RNAs. In the earliest assembly step, U1snRNA identifies the 5′ splice site. However, U1snRNA leaves the spliceosome relatively early in assembly, and 5′ splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components during the rearrangements that build the catalytic site. Using a forward genetic screen in Caenorhabditis elegans, we have identified suppressors of a locomotion defect caused by a 5′ss mutation. Here we report three new suppressor alleles from this screen, two in PRP8 and one in SNRNP200/BRR2. mRNASeq studies of these suppressor strains indicate that they also affect specific native alternative 5′ss, especially for suppressor PRP8 D1549N. A strong suppressor at the unstructured N-terminus of SNRNP200, N18K, indicates a novel role for this region. By examining distinct changes in the splicing of native genes, examining double mutants between suppressors, comparing these new suppressors to previously identified splicing suppressors from yeast, and mapping conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions at multiple stages in spliceosome assembly responsible for maintaining the initial 5′ss identified by U1snRNA for entry into the catalytic core.

    

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4. 1H, 13C, 15N resonance assignments and secondary structure of yeast oligosaccharyltransferase subunit Ost4 and its functionally important mutant Ost4V23D

   https://doi.org/10.1007/s12104-020-09946-7  

   Chaudhary, Bharat P. ; Zoetewey, David ; Mohanty, Smita ( April 2020 , Biomolecular NMR Assignments)

   Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction that occurs in the endoplasmic reticulum of cells during protein synthesis at the ribosome. In the central reaction, a pre-assembled high- mannose sugar is transferred from a lipid-linked donor substrate to the side-chain of an asparagine residue in an -N-X-T/S- sequence (where X is any residue except Proline). This reaction is carried by a membrane-bound multi-subunit enzyme complex, Oligosaccharyltransferase (OST). In humans, genetic defects in OST lead to a group of rare metabolic diseases collectively known as congenital disorders of glycosylation (CDG). Certain mutations are lethal for all organisms. In yeast, the OST is composed of nine non-identical protein subunits. The functional enzyme complex contains eight subunits with either Ost3 or Ost6 at any given time. Ost4, an unusually small protein, plays a very important role in the stabilization of the OST complex. It bridges the catalytic subunit Stt3 with Ost3 (or Ost6) in the Stt3-Ost4-Ost3 (or Ost6) sub-complex. Mutation of any residue from M18-I24 in the trans-membrane helix of yeast Ost4 negatively impacts N-linked glycosylation and the growth of yeast. Indeed, mutation of valine23 to an aspartate impairs OST function in vivo resulting in a lethal phenotype in yeast. To understand the structural mechanism of Ost4 in the stabilization of the enzyme complex, we have initiated a detailed investigation of Ost4 and its functionally important mutant, Ost4V23D. Here, we report the backbone 1H, 13C and 15N resonance assignments for Ost4 and Ost4V23D in DPC micelles. 

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5. Mutation of the polyadenylation complex subunit CstF77 reveals that mRNA 3′ end formation and HSP101 levels are critical for a robust heat stress response

   https://doi.org/10.1093/plcell/koac351  

   Kim, Minsoo ; Swenson, John ; McLoughlin, Fionn ; Vierling, Elizabeth ( December 2022 , The Plant Cell)

   Heat shock protein 101 (HSP101) in plants, and bacterial and yeast orthologs, is essential for thermotolerance. To investigate thermotolerance mechanisms involving HSP101, we performed a suppressor screen in Arabidopsis thaliana of a missense HSP101 allele (hot1–4). hot1–4 plants are sensitive to acclimation heat treatments that are otherwise permissive for HSP101 null mutants, indicating that the hot1–4 protein is toxic. We report one suppressor (shot2, suppressor of hot1–4 2) has a missense mutation of a conserved residue in CLEAVAGE STIMULATION FACTOR77 (CstF77), a subunit of the polyadenylation complex critical for mRNA 3′ end maturation. We performed ribosomal RNA depletion RNA-Seq and captured transcriptional readthrough with a custom bioinformatics pipeline. Acclimation heat treatment caused transcriptional readthrough in hot1–4 shot2, with more readthrough in heat-induced genes, reducing the levels of toxic hot1–4 protein and suppressing hot1–4 heat sensitivity. Although shot2 mutants develop like the wild type in the absence of stress and survive mild heat stress, reduction of heat-induced genes and decreased HSP accumulation makes shot2 in HSP101 null and wild-type backgrounds sensitive to severe heat stress. Our study reveals the critical function of CstF77 for 3′ end formation of mRNA and the dominant role of HSP101 in dictating the outcome of severe heat stress.

    

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