Browsing by Author "Llinas, Roxanna J."

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    A facile forward-genetic screen forﾠArabidopsisﾠautophagy mutants reveals twenty-one loss-of-function mutations disrupting sixﾠATGﾠgenes
    (Taylor & Francis, 2019) Young, Pierce G.; Passalacqua, Michael J.; Chappell, Kevin; Llinas, Roxanna J.; Bartel, Bonnie
    Macroautophagy is a process through which eukaryotic cells degrade large substrates including organelles, protein aggregates, and invading pathogens. Over 40 autophagy-related (ATG) genes have been identified through forward-genetic screens in yeast. Although homology-based analyses have identified conserved ATG genes in plants, only a few atg mutants have emerged from forward-genetic screens in Arabidopsis thaliana. We developed a screen that consistently recovers Arabidopsis atg mutations by exploiting mutants with defective LON2/At5g47040, a protease implicated in peroxisomal quality control. Arabidopsis lon2mutants exhibit reduced responsiveness to the peroxisomally-metabolized auxin precursor indole-3-butyric acid (IBA), heightened degradation of several peroxisomal matrix proteins, and impaired processing of proteins harboring N-terminal peroxisomal targeting signals; these defects are ameliorated by preventing autophagy. We optimized a lon2 suppressor screen to expedite recovery of additional atg mutants. After screening mutagenized lon2-2 seedlings for restored IBA responsiveness, we evaluated stabilization and processing of peroxisomal proteins, levels of several ATG proteins, and levels of the selective autophagy receptor NBR1/At4g24690, which accumulates when autophagy is impaired. We recovered 21 alleles disrupting 6 ATG genes: ATG2/At3g19190, ATG3/At5g61500, ATG5/At5g17290, ATG7/At5g45900, ATG16/At5g50230, and ATG18a/At3g62770. Twenty alleles were novel, and 3 of the mutated genes lack T-DNA insertional alleles in publicly available repositories. We also demonstrate that an insertional atg11/At4g30790allele incompletely suppresses lon2 defects. Finally, we show that NBR1 is not necessary for autophagy of lon2 peroxisomes and that NBR1 overexpression is not sufficient to trigger autophagy of seedling peroxisomes, indicating that Arabidopsis can use an NBR1-independent mechanism to target peroxisomes for autophagic degradation.
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    A PEX5 missense allele preferentially disrupts PTS1 cargo import into Arabidopsis peroxisomes
    (American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd., 3/20/2019) Patel, Khushali J.; Kao, Yun-Ting; Llinas, Roxanna J.; Bartel, Bonnie; BioSciences
    The sorting of eukaryotic proteins to various organellar destinations requires receptors that recognize cargo protein targeting signals and facilitate transport into the organelle. One such receptor is the peroxin PEX5, which recruits cytosolic cargo carrying a peroxisome‐targeting signal (PTS) type 1 (PTS1) for delivery into the peroxisomal lumen (matrix). In plants and mammals, PEX5 is also indirectly required for peroxisomal import of proteins carrying a PTS2 signal because PEX5 binds the PTS2 receptor, bringing the associated PTS2 cargo to the peroxisome along with PTS1 cargo. Despite PEX5 being the PTS1 cargo receptor, previously identified Arabidopsis pex5 mutants display either impairment of both PTS1 and PTS2 import or defects only in PTS2 import. Here, we report the first Arabidopsis pex5 mutant with an exclusive PTS1 import defect. In addition to markedly diminished GFP‐PTS1 import and decreased pex5‐2 protein accumulation, this pex5‐2 mutant shows typical peroxisome‐related defects, including inefficient β‐oxidation and reduced growth. Growth at reduced or elevated temperatures ameliorated or exacerbated pex5‐2 peroxisome‐related defects, respectively, without markedly changing pex5‐2 protein levels. In contrast to the diminished PTS1 import, PTS2 processing was only slightly impaired and PTS2‐GFP import appeared normal in pex5‐2. This finding suggests that even minor peroxisomal localization of the PTS1 protein DEG15, the PTS2‐processing protease, is sufficient to maintain robust PTS2 processing.
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    An Arabidopsis pre-RNA processing8a (prp8a) missense allele restores splicing of a subset of mis-spliced mRNAs
    (Oxford University Press, 2022) Llinas, Roxanna J.; Xiong, Jia Qi; Clark, Natalie M.; Burkhart, Sarah E.; Bartel, Bonnie
    Eukaryotic precursor mRNAs often harbor noncoding introns that must be removed prior to translation. Accurate splicing of precursor messenger RNA depends on placement and assembly of small nuclear ribonucleoprotein (snRNP) sub-complexes of the spliceosome. Yeast (Saccharomyces cerevisiae) studies established a role in splice-site selection for PRE-RNA PROCESSING8 (PRP8), a conserved spliceosome scaffolding protein of the U5 snRNP. However, analogous splice-site selection studies in multicellular eukaryotes are lacking. Such studies are crucial for a comprehensive understanding of alternative splicing, which is extensive in plants and animals but limited in yeast. In this work, we describe an Arabidopsis (Arabidopsis thaliana) prp8a mutant that modulates splice-site selection. We isolated prp8a-14 from a screen for suppressors of pex14-6, which carries a splice-site mutation in the PEROXIN14 (PEX14) peroxisome biogenesis gene. To elucidate Arabidopsis PRP8A function in spliceosome fidelity, we combined prp8a-14 with various pex14 splice-site mutations and monitored the double mutants for physiological and molecular consequences of dysfunctional and functional peroxisomes that correspond to impaired and recovered splicing, respectively. prp8a-14 restored splicing and PEX14 function to alleles with mutations in the exonic guanine of the 5′-splice site but did not restore splicing or function to alleles with mutations in the intronic guanine of 5′- or 3′-splice sites. We used RNA-seq to reveal the systemic impact of prp8a-14 and found hundreds of differentially spliced transcripts and thousands of transcripts with significantly altered levels. Among differentially spliced transcripts, prp8a-14 significantly altered 5′- and 3′-splice-site utilization to favor sites resulting in shorter introns. This study provides a genetic platform for probing splicing in plants and hints at a role for plant PRP8 in splice-site selection.
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    Failures in COVID-19 Vaccine Administration Data Collection Complicated Efforts to Ensure Vaccine Equity in Texas
    (2022) Laroche, Robert A.S.; Llinas, Roxanna J.; Navara, Adam M.; Tan, Melody T.; Lakshmanan, Rekha; Matthews, Kirstin R.W.; James A. Baker III Institute for Public Policy
    The equitable distribution of the COVID-19 vaccines is a critical issue due to the stark differences in health care outcomes between individuals who have received a vaccine and those who have not.1,2 Several demographic groups have historically faced barriers to accessing health care services such as vaccinations, including the elderly, rural populations, lower income groups, and communities of color.3-6 Overcoming these disadvantages and ensuring easy and affordable access are critical components of health care. Moreover, as part of an effective government response to the COVID-19 pandemic, it is necessary for members of all demographic groups to reach high rates of vaccination in order to achieve herd immunity within a region.7 In this paper, we discuss the steps taken by the Texas Department of State Health Services (TX DSHS) to ensure equitable vaccine access and to assess how shortfalls in data collection led to vaccine inequities. We recommend changes to the Texas immunization registry that will improve data collection, allow for more effective monitoring of vaccine distribution, and better prepare Texas for future public health crises.
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    Genetic suppressors reveal varying methods for improving peroxisome function in Arabidopsis peroxin mutants
    (2021-12-02) Llinas, Roxanna J.; Bartel, Bonnie; Matthews, Kathleen
    Peroxisomes are eukaryotic organelles that support several metabolic pathways critical to survival and development. These pathways include diverse oxidative reactions with harmful reactive byproducts that are neutralized with enzymes accumulated in peroxisomes. New peroxisomes can be generated via de novo formation from the endoplasmic reticulum or growth and fission of mature peroxisomes with the assistance of proteins called peroxins (PEX proteins). Once formed, peroxisomes acquire necessary enzymes by using other peroxins to import lumenal proteins. Although we have a general framework for understanding peroxisome biogenesis, we lack a complete molecular understanding of the activity and specific sequence of events through which these peroxins function. Unlike in mammals, -oxidation is exclusively peroxisomal in plants, making plants an ideal multicellular system in which to study peroxisomes and peroxisomal metabolism. Germinating Arabidopsis seedlings rely on peroxisomes to catabolize fatty acids stored in lipid droplets, and mutations in peroxin genes confer a range of easily assayed developmental defects that report peroxisome function. I elucidated molecular mechanisms underlying Arabidopsis peroxisomal biogenesis and function by characterizing peroxin mutants and peroxin mutant suppressors. These suppressors carry secondary mutations that restore peroxisomal function to Arabidopsis peroxin mutants. I examined suppressors of pex10-2 and pex12-1, mutants defective in ubiquitin-protein ligases that aid in peroxisome receptor recycling, and pex14-1 and pex14-6, mutants defective in a docking complex peroxin that aids in peroxisomal protein import. I clarified details of how these peroxins function and uncovered novel associations between peroxins by assessing the steps in peroxisomal function that are restored in the suppressors. One pex14-6 suppressor corrects the splicing defect of the original lesion, and I used it to investigate how splicing factors maintain splicing fidelity. One pex12-1 suppressor is defective in a peroxin implicated in peroxisomal membrane protein insertion and pre-peroxisome budding from the endoplasmic reticulum, and I used it to investigate a potential role in protein import. Finally, I examined the role of PEX16, an early-acting peroxisome biogenesis factor, in plants. Together, these studies revealed varying methods for modulating peroxisome function and suggest additional obscure factors in peroxisome competence.