More Like this

1. Comparative parallel analysis of RNA ends identifies mRNA substrates of a tRNA splicing endonuclease-initiated mRNA decay pathway

   https://doi.org/10.1073/pnas.2020429118  

   Hurtig, Jennifer E. ; Steiger, Michelle A. ; Nagarajan, Vinay K. ; Li, Tao ; Chao, Ti-Chun ; Tsai, Kuang-Lei ; van Hoof, Ambro ( March 2021 , Proceedings of the National Academy of Sciences)

   Eukaryotes share a conserved messenger RNA (mRNA) decay pathway in which bulk mRNA is degraded by exoribonucleases. In addition, it has become clear that more specialized mRNA decay pathways are initiated by endonucleolytic cleavage at particular sites. The transfer RNA (tRNA) splicing endonuclease (TSEN) has been studied for its ability to remove introns from pre-tRNAs. More recently it has been shown that single amino acid mutations in TSEN cause pontocerebellar hypoplasia. Other recent studies indicate that TSEN has other functions, but the nature of these functions has remained obscure. Here we show that yeast TSEN cleaves a specific subset of mRNAs that encode mitochondrial proteins, and that the cleavage sites are in part determined by their sequence. This provides an explanation for the counterintuitive mitochondrial localization of yeast TSEN. To identify these mRNA target sites, we developed a “comPARE” (comparative parallel analysis of RNA ends) bioinformatic approach that should be easily implemented and widely applicable to the study of endoribonucleases. The similarity of tRNA endonuclease-initiated decay to regulated IRE1-dependent decay of mRNA suggests that mRNA specificity by colocalization may be an important determinant for the degradation of localized mRNAs in a variety of eukaryotic cells.
2. Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

   https://doi.org/10.1038/s41467-022-31876-2  

   Arguello, A. Emilia ; Li, Ang ; Sun, Xuemeng ; Eggert, Tanner W. ; Mairhofer, Elisabeth ; Kleiner, Ralph E. ( July 2022 , Nature Communications)

   Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m⁵C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile m⁵C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm⁵C- and f⁵C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m⁵C oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure f⁵C sites on select tRNA. Finally, we show that f⁵C at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA m⁵C dioxygenases and mapping oxidative m⁵C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.
3. Agrobacterium VirE2 Protein Modulates Plant Gene Expression and Mediates Transformation From Its Location Outside the Nucleus

   https://doi.org/10.3389/fpls.2021.684192  

   Lapham, Rachelle A. ; Lee, Lan-Ying ; Xhako, Eder ; Gómez, Esteban Gañán ; Nivya, V. M. ; Gelvin, Stanton B. ( June 2021 , Frontiers in Plant Science)

   Agrobacterium effector protein VirE2 is important for plant transformation. VirE2 likely coats transferred DNA (T-DNA) in the plant cell and protects it from degradation. VirE2 localizes to the plant cytoplasm and interacts with several host proteins. Plant-expressed VirE2 can complement a virE2 mutant Agrobacterium strain to support transformation. We investigated whether VirE2 could facilitate transformation from a nuclear location by affixing to it a strong nuclear localization signal (NLS) sequence. Only cytoplasmic-, but not nuclear-localized, VirE2 could stimulate transformation. To investigate the ways VirE2 supports transformation, we generated transgenic Arabidopsis plants containing a virE2 gene under the control of an inducible promoter and performed RNA-seq and proteomic analyses before and after induction. Some differentially expressed plant genes were previously known to facilitate transformation. Knockout mutant lines of some other VirE2 differentially expressed genes showed altered transformation phenotypes. Levels of some proteins known to be important for transformation increased in response to VirE2 induction, but prior to or without induction of their corresponding mRNAs. Overexpression of some other genes whose proteins increased after VirE2 induction resulted in increased transformation susceptibility. We conclude that cytoplasmically localized VirE2 modulates both plant RNA and protein levels to facilitate transformation.
4. The Multifunctional Long-Distance Movement Protein of Pea Enation Mosaic Virus 2 Protects Viral and Host Transcripts from Nonsense-Mediated Decay

   https://doi.org/10.1128/mBio.00204-20  

   May, Jared P. ; Johnson, Philip Z. ; Ilyas, Muhammad ; Gao, Feng ; Simon, Anne E. ( April 2020 , mBio)

   Palese, Peter (Ed.)

   ABSTRACT The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3′ untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3′ UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant’s vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana , p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3′ UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor. IMPORTANCE Nonsense-mediated decaymore »(NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3′ untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.« less
5. POPEYE intercellular localization mediates cell-specific iron deficiency responses

   https://doi.org/10.1093/plphys/kiac357  

   Muhammad, DurreShahwar ; Clark, Natalie M. ; Haque, Samiul ; Williams, Cranos M. ; Sozzani, Rosangela ; Long, Terri A. ( August 2022 , Plant Physiology)

   Plants must tightly regulate iron (Fe) sensing, acquisition, transport, mobilization, and storage to ensure sufficient levels of this essential micronutrient. POPEYE (PYE) is an iron responsive transcription factor that positively regulates the iron deficiency response, while also repressing genes essential for maintaining iron homeostasis. However, little is known about how PYE plays such contradictory roles. Under iron-deficient conditions, pPYE:GFP accumulates in the root pericycle while pPYE:PYE–GFP is localized to the nucleus in all Arabidopsis (Arabidopsis thaliana) root cells, suggesting that PYE may have cell-specific dynamics and functions. Using scanning fluorescence correlation spectroscopy and cell-specific promoters, we found that PYE–GFP moves between different cells and that the tendency for movement corresponds with transcript abundance. While localization to the cortex, endodermis, and vasculature is required to manage changes in iron availability, vasculature and endodermis localization of PYE–GFP protein exacerbated pye-1 defects and elicited a host of transcriptional changes that are detrimental to iron mobilization. Our findings indicate that PYE acts as a positive regulator of iron deficiency response by regulating iron bioavailability differentially across cells, which may trigger iron uptake from the surrounding rhizosphere and impact root energy metabolism.