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1. Mitochondrial mRNA fragments are circularized in a human HEK cell line

   https://doi.org/10.1016/j.mito.2019.11.002  

   Mance, LG ; Mawla, I ; Shell, SM ; Cahoon, AB ( January 2020 , Mitochondrion)

   The relatively recent focus on the widespread occurrence and abundance of circular RNAs (circRNA) in the human cell nucleus has sparked an intensive interest in their existence and possible roles in cell gene expression and physiology. The presence of circRNAs in mammalian mitochondria, however, has been under-explored. Mitochondrial mRNAs differ from those produced from nuclear genes because they lack introns and are transcribed as poly-cistronic transcripts that are endonucleolytically cleaved, leaving transcripts with very small 5′ and 3′ UTRs. Circular RNAs have been identified in the semi-autonomous organelles of single-celled organisms and plants but their purpose has not been clearly demonstrated. The goal of our project was to test the hypothesis, processed mRNAs are circularized in vertebrate mitochondria as a necessary RNA processing step prior to translation. Mitochondrial mRNAs were isolated from the human cell line HEK293 and evidence of circularization sought by treating RNA with RNAse-R and then amplifying putative 3′-5′ junction sites. Sequence results demonstrated the occurrence of mRNA circularization within each coding region of the mitochondrial genome. However, in most cases the circRNAs carried coding regions that had been truncated, suggesting they were not translatable. Quantification of the circularized versions of the mRNAs revealed they comprise amore »small portion (~10%) of the total mRNA. These findings demonstrate that mRNA circularization occurs in mammalian mitochondria but it does not appear to play a role in making translatable mRNAs.« less
2. Beyond transcription factors: roles of mRNA decay in regulating gene expression in plants

   https://doi.org/10.12688/f1000research.16203.1  

   Sieburth, Leslie E ; Vincent, Jessica N ( January 2018 , F1000Research)

   Gene expression is typically quantified as RNA abundance, which is influenced by both synthesis (transcription) and decay. Cytoplasmic decay typically initiates by deadenylation, after which decay can occur through any of three cytoplasmic decay pathways. Recent advances reveal several mechanisms by which RNA decay is regulated to control RNA abundance. mRNA can be post-transcriptionally modified, either indirectly through secondary structure or through direct modifications to the transcript itself, sometimes resulting in subsequent changes in mRNA decay rates. mRNA abundances can also be modified by tapping into pathways normally used for RNA quality control. Regulated mRNA decay can also come about through post-translational modification of decapping complex subunits. Likewise, mRNAs can undergo changes in subcellular localization (for example, the deposition of specific mRNAs into processing bodies, or P-bodies, where stabilization and destabilization occur in a transcript- and context-dependent manner). Additionally, specialized functions of mRNA decay pathways were implicated in a genome-wide mRNA decay analysis in Arabidopsis. Advances made using plants are emphasized in this review, but relevant studies from other model systems that highlight RNA decay mechanisms that may also be conserved in plants are discussed.
3. Mitochondrial mRNA Processing in the Chlorophyte Alga Pediastrum duplex and Streptophyte Alga Chara vulgaris Reveals an Evolutionary Branch in Mitochondrial mRNA Processing

   https://doi.org/10.3390/plants10030576  

   Proulex, Grayson C. ; Meade, Marcus J. ; Manoylov, Kalina M. ; Cahoon, A. Bruce ( March 2021 , Plants)

   Mitochondria carry the remnant of an ancestral bacterial chromosome and express those genes with a system separate and distinct from the nucleus. Mitochondrial genes are transcribed as poly-cistronic primary transcripts which are post-transcriptionally processed to create individual translationally competent mRNAs. Algae post-transcriptional processing has only been explored in Chlamydomonas reinhardtii (Class: Chlorophyceae) and the mature mRNAs are different than higher plants, having no 5′ UnTranslated Regions (UTRs), much shorter and more variable 3′ UTRs and polycytidylated mature mRNAs. In this study, we analyzed transcript termini using circular RT-PCR and PacBio Iso-Seq to survey the 3′ and 5′ UTRs and termini for two green algae, Pediastrum duplex (Class: Chlorophyceae) and Chara vulgaris (Class: Charophyceae). This enabled the comparison of processing in the chlorophyte and charophyte clades of green algae to determine if the differences in mitochondrial mRNA processing pre-date the invasion of land by embryophytes. We report that the 5′ mRNA termini and non-template 3′ termini additions in P. duplex resemble those of C. reinhardtii, suggesting a conservation of mRNA processing among the chlorophyceae. We also report that C. vulgaris mRNA UTRs are much longer than chlorophytic examples, lack polycytidylation, and are polyadenylated similar to embryophytes. This demonstrates that some mitochondrialmore »mRNA processing events diverged with the split between chlorophytic and streptophytic algae.« less
4. Ubiquitous mRNA decay fragments in E. coli redefine the functional transcriptome

   https://doi.org/10.1093/nar/gkac295  

   Herzel, Lydia ; Stanley, Julian A. ; Yao, Chun-Chen ; Li, Gene-Wei ( May 2022 , Nucleic Acids Research)

   Bacterial mRNAs have short life cycles, in which transcription is rapidly followed by translation and degradation within seconds to minutes. The resulting diversity of mRNA molecules across different life-cycle stages impacts their functionality but has remained unresolved. Here we quantitatively map the 3’ status of cellular RNAs in Escherichia coli during steady-state growth and report a large fraction of molecules (median>60%) that are fragments of canonical full-length mRNAs. The majority of RNA fragments are decay intermediates, whereas nascent RNAs contribute to a smaller fraction. Despite the prevalence of decay intermediates in total cellular RNA, these intermediates are underrepresented in the pool of ribosome-associated transcripts and can thus distort quantifications and differential expression analyses for the abundance of full-length, functional mRNAs. The large heterogeneity within mRNA molecules in vivo highlights the importance in discerning functional transcripts and provides a lens for studying the dynamic life cycle of mRNAs.
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 ofmore »severe heat stress.

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