Kevin Richard Jones Roy

All Publications

• Splicing factor Prp18p promotes genome-wide fidelity of consensus 3'-splice sites NUCLEIC ACIDS RESEARCH Roy, K. R., Gabunilas, J., Neutel, D., Ai, M., Yeh, Z., Samson, J., Lyu, G., Chanfreau, G. F. 2023

  Abstract

  The fidelity of splice site selection is critical for proper gene expression. In particular, proper recognition of 3'-splice site (3'SS) sequences by the spliceosome is challenging considering the low complexity of the 3'SS consensus sequence YAG. Here, we show that absence of the Prp18p splicing factor results in genome-wide activation of alternative 3'SS in S. cerevisiae, including highly unusual non-YAG sequences. Usage of these non-canonical 3'SS in the absence of Prp18p is enhanced by upstream poly(U) tracts and by their potential to interact with the first intronic nucleoside, allowing them to dock in the spliceosome active site instead of the normal 3'SS. The role of Prp18p in 3'SS fidelity is facilitated by interactions with Slu7p and Prp8p, but cannot be fulfilled by Slu7p, identifying a unique role for Prp18p in 3'SS fidelity. This fidelity function is synergized by the downstream proofreading activity of the Prp22p helicase, but is independent from another late splicing helicase, Prp43p. Our results show that spliceosomes exhibit remarkably relaxed 3'SS sequence usage in the absence of Prp18p and identify a network of spliceosomal interactions centered on Prp18p which are required to promote the fidelity of the recognition of consensus 3'SS sequences.

  View details for DOI 10.1093/nar/gkad968

  View details for Web of Science ID 001101747800001

  View details for PubMedID 37956322

• CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and Bioinformatics Laboratory Module for Undergraduate and High School Students. Journal of microbiology & biology education Sankaran, S. M., Smith, J. D., Roy, K. R. 2021; 22 (2)

  Abstract

  CRISPR-Cas9 genome editing technology is widely used in scientific research and biotechnology. As this technology becomes a staple tool in life sciences research, it is increasingly important to incorporate it into biology curricula to train future scientists. To demonstrate the molecular underpinnings and some limitations of CRISPR-based gene editing, we designed a laboratory module to accompany a discussion-based course on genome editing for college and advanced high school biology students. The laboratory module uses CRISPR-Cas9 to target and inactivate the ADE2 gene in Saccharomyces cerevisiae so as to give red colonies, employing an inexpensive yeast model system with a phenotypic readout that is easily detectable without specialized equipment. Students begin by accessing the yeast ADE2 sequence in a genome database, applying their understanding of Cas9 activity to design guide RNA (gRNA) sequences, using a CRISPR analysis tool to compare predicted on- and off-target effects of various gRNAs, and presenting and explaining their choice of an optimal gRNA to disrupt the ADE2 gene. They then conduct yeast transformations using Cas9 and preselected gRNA plasmids with or without donor templates to explore the importance of DNA repair pathways in genome editing. Lastly, they analyze the observed editing rates across different gRNAs targeting ADE2, leading to a discussion of editing efficiency. This module engages students in experimental design, provides hands-on experience with CRISPR-Cas9 gene editing and collaborative data analysis, and stimulates discussion on the uses and limitations of CRISPR-based gene editing technology.

  View details for DOI 10.1128/jmbe.00106-21

  View details for PubMedID 34594460

• Multiplexed precision genome editing with trackable genomic barcodes in yeast. Nature biotechnology Roy, K. R., Smith, J. D., Vonesch, S. C., Lin, G., Tu, C. S., Lederer, A. R., Chu, A., Suresh, S., Nguyen, M., Horecka, J., Tripathi, A., Burnett, W. T., Morgan, M. A., Schulz, J., Orsley, K. M., Wei, W., Aiyar, R. S., Davis, R. W., Bankaitis, V. A., Haber, J. E., Salit, M. L., St Onge, R. P., Steinmetz, L. M. 2018

  Abstract

  Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here we describe a CRISPR-Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide-donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA-Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.

  View details for PubMedID 29734294

• Common genomic elements promote transcriptional and DNA replication roadblocks. Genome research Roy, K., Gabunilas, J., Gillespie, A., Ngo, D., Chanfreau, G. F. 2016; 26 (10): 1363-1375

  Abstract

  RNA polymerase II (Pol II) transcription termination by the Nrd1p-Nab3p-Sen1p (NNS) pathway is critical for the production of stable noncoding RNAs and the control of pervasive transcription in Saccharomyces cerevisiae To uncover determinants of NNS termination, we mapped the 3'-ends of NNS-terminated transcripts genome-wide. We found that nucleosomes and specific DNA-binding proteins, including the general regulatory factors (GRFs) Reb1p, Rap1p, and Abf1p, and Pol III transcription factors enhance the efficiency of NNS termination by physically blocking Pol II progression. The same DNA-bound factors that promote NNS termination were shown previously to define the 3'-ends of Okazaki fragments synthesized by Pol δ during DNA replication. Reduced binding of these factors results in defective NNS termination and Pol II readthrough. Furthermore, inactivating NNS enables Pol II elongation through these roadblocks, demonstrating that effective Pol II termination depends on a synergy between the NNS machinery and obstacles in chromatin. Consistent with this finding, loci exhibiting Pol II readthrough at GRF binding sites are depleted for upstream NNS signals. Overall, these results underscore how RNA termination signals influence the behavior of Pol II at chromatin obstacles, and establish that common genomic elements define boundaries for both DNA and RNA synthesis machineries.

  View details for DOI 10.1101/gr.204776.116

  View details for PubMedID 27540088

  View details for PubMedCentralID PMC5052057

• Stress-Induced Nuclear RNA Degradation Pathways Regulate Yeast Bromodomain Factor 2 to Promote Cell Survival PLOS GENETICS Roy, K., Chanfreau, G. 2014; 10 (9)

  Abstract

  Bromodomain proteins are key regulators of gene expression. How the levels of these factors are regulated in specific environmental conditions is unknown. Previous work has established that expression of yeast Bromodomain factor 2 (BDF2) is limited by spliceosome-mediated decay (SMD). Here we show that BDF2 is subject to an additional layer of post-transcriptional control through RNase III-mediated decay (RMD). We found that the yeast RNase III Rnt1p cleaves a stem-loop structure within the BDF2 mRNA to down-regulate its expression. However, these two nuclear RNA degradation pathways play distinct roles in the regulation of BDF2 expression, as we show that the RMD and SMD pathways of the BDF2 mRNA are differentially activated or repressed in specific environmental conditions. RMD is hyper-activated by salt stress and repressed by hydroxyurea-induced DNA damage while SMD is inactivated by salt stress and predominates during DNA damage. Mutations of cis-acting signals that control SMD and RMD rescue numerous growth defects of cells lacking Bdf1p, and show that SMD plays an important role in the DNA damage response. These results demonstrate that specific environmental conditions modulate nuclear RNA degradation pathways to control BDF2 expression and Bdf2p-mediated gene regulation. Moreover, these results show that precise dosage of Bromodomain factors is essential for cell survival in specific environmental conditions, emphasizing their importance for controlling chromatin structure and gene expression in response to environmental stress.

  View details for DOI 10.1371/journal.pgen.1004661

  View details for Web of Science ID 000343009600058

  View details for PubMedID 25232960

• Genome-scale analysis of interactions between genetic perturbations and natural variation. bioRxiv : the preprint server for biology Hale, J. J., Matsui, T., Goldstein, I., Mullis, M. N., Roy, K. R., Ville, C. N., Miller, D., Wang, C., Reynolds, T., Steinmetz, L. M., Levy, S. F., Ehrenreich, I. M. 2024

  Abstract

  Interactions between genetic perturbations and segregating loci can cause perturbations to show different phenotypic effects across genetically distinct individuals. To study these interactions on a genome scale in many individuals, we used combinatorial DNA barcode sequencing to measure the fitness effects of 7,700 CRISPRi perturbations targeting 1,712 distinct genes in 169 yeast cross progeny (or segregants). We identified 460 genes whose perturbation has different effects across segregants. Several factors caused perturbations to show variable effects, including baseline segregant fitness, the mean effect of a perturbation across segregants, and interacting loci. We mapped 234 interacting loci and found four hub loci that interact with many different perturbations. Perturbations that interact with a given hub exhibit similar epistatic relationships with the hub and show enrichment for cellular processes that may mediate these interactions. These results suggest that an individual's response to perturbations is shaped by a network of perturbation-locus interactions that cannot be measured by approaches that examine perturbations or natural variation alone.

  View details for DOI 10.1101/2023.05.06.539663

  View details for PubMedID 38293072

  View details for PubMedCentralID PMC10827069

• Large scale microfluidic CRISPR screening for increased amylase secretion in yeast. Lab on a chip Johansson, S. A., Dulermo, T., Jann, C., Smith, J. D., Pryszlak, A., Pignede, G., Schraivogel, D., Colavizza, D., Desfougères, T., Rave, C., Farwick, A., Merten, C. A., Roy, K. R., Wei, W., Steinmetz, L. M. 2023

  Abstract

  Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

  View details for DOI 10.1039/d3lc00111c

  View details for PubMedID 37483015

• A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa. bioRxiv : the preprint server for biology Neumayer, G., Torkelson, J. L., Li, S., McCarthy, K., Zhen, H. H., Vangipuram, M., Jackow, J., Rami, A., Hansen, C., Guo, Z., Gaddam, S., Pappalardo, A., Li, L., Cramer, A., Roy, K. R., Nguyen, T. M., Tanabe, K., McGrath, P. S., Bruckner, A., Bilousova, G., Roop, D., Bailey, I., Tang, J. Y., Christiano, A., Steinmetz, L. M., Wernig, M., Oro, A. E. 2023

  Abstract

  Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies.We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing.iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice.DEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

  View details for DOI 10.1101/2023.02.28.529447

  View details for PubMedID 36909618

  View details for PubMedCentralID PMC10002612

• The interplay of additivity, dominance, and epistasis on fitness in a diploid yeast cross. Nature communications Matsui, T., Mullis, M. N., Roy, K. R., Hale, J. J., Schell, R., Levy, S. F., Ehrenreich, I. M. 2022; 13 (1): 1463

  Abstract

  In diploid species, genetic loci can show additive, dominance, and epistatic effects. To characterize the contributions of these different types of genetic effects to heritable traits, we use a double barcoding system to generate and phenotype a panel of ~200,000 diploid yeast strains that can be partitioned into hundreds of interrelated families. This experiment enables the detection of thousands of epistatic loci, many whose effects vary across families. Here, we show traits are largely specified by a small number of hub loci with major additive and dominance effects, and pervasive epistasis. Genetic background commonly influences both the additive and dominance effects of loci, with multiple modifiers typically involved. The most prominent dominance modifier in our data is the mating locus, which has no effect on its own. Our findings show that the interplay between additivity, dominance, and epistasis underlies a complex genotype-to-phenotype map in diploids.

  View details for DOI 10.1038/s41467-022-29111-z

  View details for PubMedID 35304450

• Engineered Graphene Oxide Nanocomposite Capable of Preventing the Evolution of Antimicrobial Resistance. ACS nano Zheng, H. n., Ji, Z. n., Roy, K. R., Gao, M. n., Pan, Y. n., Cai, X. n., Wang, L. n., Li, W. n., Chang, C. H., Kaweeteerawat, C. n., Chen, C. n., Xia, T. n., Zhao, Y. n., Li, R. n. 2019

  Abstract

  Antimicrobial resistance (AMR) is spreading worldwide and keeps evolving to adapt to antibiotics, causing increasing threats in clinics, which necessitates the exploration of antimicrobial agents for not only killing of resistant cells but also prevention of AMR progression. However, so far, there has been no effective approach. Herein, we designed lanthanum hydroxide and graphene oxide nanocomposites (La@GO) to confer a synergistic bactericidal effect in all tested resistant strains. More importantly, long-term exposure of E. coli (AMR) to subminimum inhibitory concentrations of La@GO does not trigger detectable secondary resistance, while conventional antibiotics and silver nanoparticles lead to a 16- to 64-fold increase in tolerance. The inability of E. coli to evolve resistance to La@GO is likely due to a distinctive extracellular multitarget invasion killing mechanism involving lipid dephosphorylation, lipid peroxidation, and peptidoglycan disruption. Overall, our results highlight La@GO nanocomposites as a promising solution to combating resistant bacteria without inducing the evolution of AMR.

  View details for DOI 10.1021/acsnano.9b04970

  View details for PubMedID 31566947

• Robust mapping of polyadenylated and non-polyadenylated RNA 3' ends at nucleotide resolution by 3'-end sequencing. Methods (San Diego, Calif.) Roy, K. R., Chanfreau, G. F. 2019

  Abstract

  3'-end poly(A)+ sequencing is an efficient and economical method for global measurement of mRNA levels and alternative poly(A) site usage. A common method involves oligo(dT)19V reverse-transcription (RT)-based library preparation and high-throughput sequencing with a custom primer ending in (dT)19. While the majority of library products have the first sequenced nucleotide reflect the bona fide poly(A) site (pA), a substantial fraction of sequencing reads arise from various mis-priming events. These can result in incorrect pA site calls anywhere from several nucleotides downstream to several kilobases upstream from the bona fide pA site. While these mis-priming events can be mitigated by increasing annealing stringency (e.g. increasing temperature from 37 °C to 42 °C), they still persist at an appreciable level (∼10%) and computational methods must be used to prevent artifactual calls. Here we present a bioinformatics workflow for precise mapping of poly(A)+ 3' ends and handling of artifacts due to oligo(dT) mis-priming and sample polymorphisms. We test pA site calling with three different read mapping programs (STAR, BWA, and BBMap), and show that the way in which each handles terminal mismatches and soft clipping has a substantial impact on identifying correct pA sites, with BWA requiring the least post-processing to correct artifacts. We demonstrate the use of this pipeline for mapping pA sites in the model eukaryote S. cerevisiae, and further apply this technology to non-polyadenylated transcripts by employing in vitro polyadenylation prior to library prep (IVP-seq). As proof of principle, we show that a fraction of tRNAs harbor CCU 3' tails instead of the canonical CCA tail, and globally identify 3' ends of splicing intermediates arising from inefficiently spliced transcripts.

  View details for DOI 10.1016/j.ymeth.2019.05.016

  View details for PubMedID 31128237

• A global function for transcription factors in assisting RNA polymerase II termination. Transcription Roy, K., Chanfreau, G. F. 2018; 9 (1): 41-46

  Abstract

  The role of transcription factors (TFs) on nucleosome positioning, RNA polymerase recruitment, and transcription initiation has been extensively characterized. Here, we propose that a subset of TFs such as Reb1, Abf1, Rap1, and TFIIIB also serve a major function in partitioning transcription units by assisting the Nrd1p-Nab3p-Sen1p Pol II termination pathway.

  View details for DOI 10.1080/21541264.2017.1300121

  View details for PubMedID 29106321

  View details for PubMedCentralID PMC5791854

• Multiplexed precision genome editing with trackable genomic barcodes in yeast NATURE BIOTECHNOLOGY Roy, K. R., Smith, J. D., Vonesch, S. C., Lin, G., Tu, C., Lederer, A. R., Chu, A., Suresh, S., Nguyen, M., Horecka, J., Tripathi, A., Burnett, W. T., Morgan, M. A., Schulz, J., Orsley, K. M., Wei, W., Aiyar, R. S., Davis, R. W., Bankaitis, V. A., Haber, J. E., Salit, M. L., St Onge, R. P., Steinmetz, L. M. 2018; 36 (6): 512-+

  View details for DOI 10.1038/nbt.4137

  View details for Web of Science ID 000434689200019

• A method for high-throughput production of sequence-verified DNA libraries and strain collections. Molecular systems biology Smith, J. D., Schlecht, U., Xu, W., Suresh, S., Horecka, J., Proctor, M. J., Aiyar, R. S., Bennett, R. A., Chu, A., Li, Y. F., Roy, K., Davis, R. W., Steinmetz, L. M., Hyman, R. W., Levy, S. F., St Onge, R. P. 2017; 13 (2): 913-?

  Abstract

  The low costs of array-synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost-effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site-specific recombination to index library DNA, and next-generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost-effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.

  View details for DOI 10.15252/msb.20167233

  View details for PubMedID 28193641

  View details for PubMedCentralID PMC5327727

• Methylation of yeast ribosomal protein Rpl3 promotes translational elongation fidelity. RNA (New York, N.Y.) Al-Hadid, Q., Roy, K., Chanfreau, G., Clarke, S. G. 2016; 22 (4): 489-498

  Abstract

  Rpl3, a highly conserved ribosomal protein, is methylated at histidine 243 by the Hpm1 methyltransferase in Saccharomyces cerevisiae. Histidine 243 lies close to the peptidyl transferase center in a functionally important region of Rpl3 designated as the basic thumb that coordinates the decoding, peptidyl transfer, and translocation steps of translation elongation. Hpm1 was recently implicated in ribosome biogenesis and translation. However, the biological role of methylation of its Rpl3 substrate has not been identified. Here we interrogate the role of Rpl3 methylation at H243 by investigating the functional impact of mutating this histidine residue to alanine (rpl3-H243A). Akin to Hpm1-deficient cells, rpl3-H243A cells accumulate 35S and 23S pre-rRNA precursors to a similar extent, confirming an important role for histidine methylation in pre-rRNA processing. In contrast, Hpm1-deficient cells but not rpl3-H243A mutants show perturbed levels of ribosomal subunits. We show that Hpm1 has multiple substrates in different subcellular fractions, suggesting that methylation of proteins other than Rpl3 may be important for controlling ribosomal subunit levels. Finally, translational fidelity assays demonstrate that like Hpm1-deficient cells, rpl3-H243A mutants have defects in translation elongation resulting in decreased translational accuracy. These data suggest that Rpl3 methylation at H243 is playing a significant role in translation elongation, likely via the basic thumb, but has little impact on ribosomal subunit levels. Hpm1 is therefore a multifunctional methyltransferase with independent roles in ribosome biogenesis and translation.

  View details for DOI 10.1261/rna.054569.115

  View details for PubMedID 26826131

• Cu Nanoparticles Have Different Impacts in Escherichia coli and Lactobacillus brevis than Their Microsized and Ionic Analogues ACS NANO Kaweeteerawat, C., Chang, C. H., Roy, K. R., Liu, R., Li, R., Toso, D., Fischer, H., Ivask, A., Ji, Z., Zink, J. I., Zhou, Z. H., Chanfreau, G. F., Telesca, D., Cohen, Y., Holden, P. A., Nel, A. E., Godwin, H. A. 2015; 9 (7): 7215-7225

  Abstract

  Copper formulations have been used for decades for antimicrobial and antifouling applications. With the development of nanoformulations of copper that are more effective than their ionic and microsized analogues, a key regulatory question is whether these materials should be treated as new or existing materials. To address this issue, here we compare the magnitude and mechanisms of toxicity of a series of Cu species (at concentration ranging from 2 to 250 μg/mL), including nano Cu, nano CuO, nano Cu(OH)2 (CuPro and Kocide), micro Cu, micro CuO, ionic Cu(2+) (CuCl2 and CuSO4) in two species of bacteria (Escherichia coli and Lactobacillus brevis). The primary size of the particles studied ranged from 10 nm to 10 μm. Our results reveal that Cu and CuO nanoparticles (NPs) are more toxic than their microsized counterparts at the same Cu concentration, with toxicities approaching those of the ionic Cu species. Strikingly, these NPs showed distinct differences in their mode of toxicity when compared to the ionic and microsized Cu, highlighting the unique toxicity properties of materials at the nanoscale. In vitro DNA damage assays reveal that both nano Cu and microsized Cu are capable of causing complete degradation of plasmid DNA, but electron tomography results show that only nanoformulations of Cu are internalized as intact intracellular particles. These studies suggest that nano Cu at the concentration of 50 μg/mL may have unique genotoxicity in bacteria compared to ionic and microsized Cu.

  View details for DOI 10.1021/acsnano.5b02021

  View details for Web of Science ID 000358823200058

  View details for PubMedID 26168153

• Translational Roles of Elongation Factor 2 Protein Lysine Methylation JOURNAL OF BIOLOGICAL CHEMISTRY Dzialo, M. C., Travaglini, K. J., Shen, S., Roy, K., Chanfreau, G. F., Loo, J. A., Clarke, S. G. 2014; 289 (44): 30511-30524

  Abstract

  Methylation of various components of the translational machinery has been shown to globally affect protein synthesis. Little is currently known about the role of lysine methylation on elongation factors. Here we show that in Saccharomyces cerevisiae, the product of the EFM3/YJR129C gene is responsible for the trimethylation of lysine 509 on elongation factor 2. Deletion of EFM3 or of the previously described EFM2 increases sensitivity to antibiotics that target translation and decreases translational fidelity. Furthermore, the amino acid sequences of Efm3 and Efm2, as well as their respective methylation sites on EF2, are conserved in other eukaryotes. These results suggest the importance of lysine methylation modification of EF2 in fine tuning the translational apparatus.

  View details for DOI 10.1074/jbc.M114.605527

  View details for Web of Science ID 000344549700030

  View details for PubMedID 25231983

  View details for PubMedCentralID PMC4215232

• Histidine methylation of yeast ribosomal protein Rpl3p is required for proper 60S subunit assembly. Molecular and cellular biology Al-Hadid, Q., Roy, K., Munroe, W., Dzialo, M. C., Chanfreau, G. F., Clarke, S. G. 2014; 34 (15): 2903-16

  Abstract

  Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the large ribosomal subunit protein Rpl3p is methylated at histidine 243, a residue that contacts the 25S rRNA near the P site. Rpl3p methylation is dependent upon the presence of Hpm1p, a candidate seven-beta-strand methyltransferase. In this study, we elucidated the biological activities of Hpm1p in vitro and in vivo. Amino acid analyses reveal that Hpm1p is responsible for all of the detectable protein histidine methylation in yeast. The modification is found on a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nucleus-containing organelle fraction but was not detected in proteins of the ribosome-free cytosol fraction. In vitro assays demonstrate that Hpm1p has methyltransferase activity on ribosome-associated but not free Rpl3p, suggesting that its activity depends on interactions with ribosomal components. hpm1 null cells are defective in early rRNA processing, resulting in a deficiency of 60S subunits and translation initiation defects that are exacerbated in minimal medium. Cells lacking Hpm1p are resistant to cycloheximide and verrucarin A and have decreased translational fidelity. We propose that Hpm1p plays a role in the orchestration of the early assembly of the large ribosomal subunit and in faithful protein production.

  View details for DOI 10.1128/MCB.01634-13

  View details for PubMedID 24865971

  View details for PubMedCentralID PMC4135575

• Intrinsic Dynamics of an Extended Hydrophobic Core in the S. cerevisiae RNase III dsRBD Contributes to Recognition of Specific RNA Binding Sites JOURNAL OF MOLECULAR BIOLOGY Hartman, E., Wang, Z., Zhang, Q., Roy, K., Chanfreau, G., Feigon, J. 2013; 425 (3): 546-562

  Abstract

  The Saccharomyces cerevisiae RNase III enzyme Rnt1p preferentially binds to double-stranded RNA hairpin substrates with a conserved (A/u)GNN tetraloop fold, via shape-specific interactions by its double-stranded RNA-binding domain (dsRBD) helix α1 to the tetraloop minor groove. To investigate whether conformational flexibility in the dsRBD regulates the binding specificity, we determined the backbone dynamics of the Rnt1p dsRBD in the free and AGAA hairpin-bound states using NMR spin-relaxation experiments. The intrinsic microsecond-to-millisecond timescale dynamics of the dsRBD suggests that helix α1 undergoes conformational sampling in the free state, with large dynamics at some residues in the α1-β1 loop (α1-β1 hinge). To correlate free dsRBD dynamics with structural changes upon binding, we determined the solution structure of the free dsRBD used in the previously determined RNA-bound structures. The Rnt1p dsRBD has an extended hydrophobic core comprising helix α1, the α1-β1 loop, and helix α3. Analysis of the backbone dynamics and structures of the free and bound dsRBD reveals that slow-timescale dynamics in the α1-β1 hinge are associated with concerted structural changes in the extended hydrophobic core that govern binding of helix α1 to AGAA tetraloops. The dynamic behavior of the dsRBD bound to a longer AGAA hairpin reveals that dynamics within the hydrophobic core differentiate between specific and nonspecific sites. Mutations of residues in the α1-β1 hinge result in changes to the dsRBD stability and RNA-binding affinity and cause defects in small nucleolar RNA processing invivo. These results reveal that dynamics in the extended hydrophobic core are important for binding site selection by the Rnt1p dsRBD.

  View details for DOI 10.1016/j.jmb.2012.11.025

  View details for Web of Science ID 000315308900009

  View details for PubMedID 23201338

• The Diverse Functions of Fungal RNase III Enzymes in RNA Metabolism. The Enzymes Roy, K., Chanfreau, G. F. 2012; 31: 213-35

  Abstract

  Enzymes from the ribonuclease III family bind and cleave double-stranded RNA to initiate RNA processing and degradation of a large number of transcripts in bacteria and eukaryotes. This chapter focuses on the description of the diverse functions of fungal RNase III members in the processing and degradation of cellular RNAs, with a particular emphasis on the well-characterized representative in Saccharomyces cerevisiae, Rnt1p. RNase III enzymes fulfill important functions in the processing of the precursors of various stable noncoding RNAs such as ribosomal RNAs and small nuclear and nucleolar RNAs. In addition, they cleave and promote the degradation of specific mRNAs or improperly processed forms of certain mRNAs. The cleavage of these mRNAs serves both surveillance and regulatory functions. Finally, recent advances have shown that RNase III enzymes are involved in mediating fail-safe transcription termination by RNA polymerase II (Pol II), by cleaving intergenic stem-loop structures present downstream from Pol II transcription units. Many of these processing functions appear to be conserved in fungal species close to the Saccharomyces genus, and even in more distant eukaryotic species.

  View details for DOI 10.1016/B978-0-12-404740-2.00010-0

  View details for PubMedID 27166447

• Structure of a Yeast RNase III dsRBD Complex with a Noncanonical RNA Substrate Provides New Insights into Binding Specificity of dsRBDs STRUCTURE Wang, Z., Hartman, E., Roy, K., Chanfreau, G., Feigon, J. 2011; 19 (7): 999-1010

  Abstract

  dsRBDs often bind dsRNAs with some specificity, yet the basis for this is poorly understood. Rnt1p, the major RNase III in Saccharomyces cerevisiae, cleaves RNA substrates containing hairpins capped by A/uGNN tetraloops, using its dsRBD to recognize a conserved tetraloop fold. However, the identification of a Rnt1p substrate with an AAGU tetraloop raised the question of whether Rnt1p binds to this noncanonical substrate differently than to A/uGNN tetraloops. The solution structure of Rnt1p dsRBD bound to an AAGU-capped hairpin reveals that the tetraloop undergoes a structural rearrangement upon binding to Rnt1p dsRBD to adopt a backbone conformation that is essentially the same as the AGAA tetraloop, and indicates that a conserved recognition mode is used for all Rnt1p substrates. Comparison of free and RNA-bound Rnt1p dsRBD reveals that tetraloop-specific binding requires a conformational change in helix α1. Our findings provide a unified model of binding site selection by this dsRBD.

  View details for DOI 10.1016/j.str.2011.03.022

  View details for Web of Science ID 000292789300012

  View details for PubMedID 21742266