Biology subjects

Read, L. K.

Publications and source records attributed to Read, L. K..

4 recordsLinked to original sources

bioRxiv2023

Translational control by Trypanosoma brucei DRBD18 contributes to the maintenance of the procyclic state

Trypanosoma brucei occupies distinct niches throughout its life cycle, within both the mammalian and tsetse fly hosts. The immunological and biochemical complexity and variability of each of these environments require a reshaping of the protein landscape of the parasite both to evade surveillance and face changing metabolic demands. Whereas most well-studied organisms rely on transcriptional control as the main regulator of gene expression, post-transcriptional control mechanisms are particularly important in T. brucei, and these are often mediated by RNA-binding proteins. DRBD18 is a T. brucei RNA-binding protein that interacts with ribosomal proteins and translation factors. Here, we tested a role for DRBD18 in translational control. We show that DRBD18 depletion by RNA interference leads to altered polysomal profiles with a specific depletion of heavy polysomes. Ribosome profiling analysis reveals that 101 transcripts change in translational efficiency (TE) upon DRBD18 depletion: 41 exhibit decreased TE and 60 exhibit increased TE. A further 66 transcripts are buffered, i.e. changes in transcript abundance are compensated by changes in TE such that the total translational output is expected not to change. Proteomic analysis validates these data. In DRBD18-depleted cells, a cohort of transcripts that codes for procyclic form-specific proteins is translationally repressed while, conversely, transcripts that code for bloodstream form- and metacyclic form-specific proteins are translationally enhanced. These data suggest that DRBD18 contributes to the maintenance of the procyclic state through both positive and negative translational control of specific mRNAs.

microbiology↗

bioRxiv2022

Conserved and transcript-specific functions of the RESC factors, RESC13 and RESC14, in kinetoplastid RNA editing

Uridine insertion/deletion RNA editing is an extensive post-transcriptional modification of mitochondrial mRNAs in kinetoplastid organisms, including Trypanosoma brucei. This process is carried out using trans-acting gRNAs and complex protein machinery. The essential RNA Editing Substrate Binding Complex (RESC) serves as the scaffold that modulates protein and RNA interactions during editing, and contains the Guide RNA Binding Complex (GRBC), the RNA Editing Mediator Complexes (REMCs), and organizer proteins. Despite the importance of RESC in editing, the functions of each protein comprising this complex are not completely understood. Here, we further define the roles of a REMC protein, RESC13, and a RESC organizer, RESC14, using high-throughput sequencing on two large pan-edited mRNAs, A6 and COIII. When comparing our analyses to that of a previously published small pan-edited mRNA, RPS12, we find that RESC13 has conserved functions across the three transcripts with regards to editing initiation, gRNA utilization, gRNA exchange, and restricting the formation of long mis-edited junctions that likely arise from its ability to modulate RNA structure. However, RESC13 does have transcript-specific effects on the types of long junctions whose formation it restricts. RESC14 has a conserved effect on gRNA utilization across the three transcripts analyzed, but has transcript-specific effects on editing initiation, gRNA exchange, and junction formation. Our data suggest that transcript-specific effects of both proteins are due to differences in transcript length and sequences as well as transcript-specific protein interactions. These findings highlight the importance of studying multiple transcripts to determine the function of editing factors.

molecular biology↗

bioRxiv2021

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

microbiology↗

bioRxiv2019

Structural Basis of Protein Arginine Methyltransferase Activation by a Catalytically Dead Homolog (Prozyme)

Prozymes are pseudoenzymes that stimulate the function of weakly active enzymes through complex formation. The major Trypanosoma brucei protein arginine methyltransferase, TbPRMT1 enzyme (ENZ), requires TbPRMT1 prozyme (PRO) to form an active heterotetrameric complex. Here we present the x-ray crystal structure of the ENZ-{Delta}52PRO tetrameric complex with the cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.4 [A] resolution. The individual ENZ and PRO units adopt the highly-conserved PRMT domain architecture and form an antiparallel heterodimer that corresponds to the canonical homodimer observed in all previously reported PRMTs. In turn, two such heterodimers assemble into a tetramer both in the crystal and in solution with twofold rotational symmetry. ENZ is unstable in absence of PRO and incapable of forming a homodimer due to a steric clash of a non-conserved tyrosine within the dimerization arm, rationalizing why PRO is required to complement ENZ to form a PRMT dimer that is necessary, but not sufficient for PRMT activity. The PRO structure deviates from other, active PRTMs in that it lacks the conserved {eta}2 310-helix within the Rossmann fold, abolishing co-factor binding. In addition to its chaperone function for ENZ, PRO substantially contributes to substrate binding. Heterotetramerization is required for catalysis, since heterodimeric ENZ-PRO mutants lack binding affinity and methyltransferase activity towards the substrate protein TbRGG1. Together, we provide a structural basis for PRMT1 ENZ activation by PRO heterotetramer formation, which is conserved across all kinetoplastids, and describe a chaperone function of the PRMT1 prozyme, which represents a novel mode of PRMT regulation.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC=\"FIGDIR/small/776112v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (56K):\norg.highwire.dtl.DTLVardef@3d970dorg.highwire.dtl.DTLVardef@187f03forg.highwire.dtl.DTLVardef@1adee39org.highwire.dtl.DTLVardef@1f30f34_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LIThe crystal structure of Trypanosoma brucei protein arginine methyltransferase 1 (PRMT1) is reported\nC_LIO_LITwo enzyme-prozyme heterodimers form a stable heterotetramer essential for catalysis\nC_LIO_LIThe catalytically dead prozyme lacks elements essential for AdoMet binding\nC_LIO_LIThe enzyme alone is unstable and cannot form a canonical dimer due to steric clashes\nC_LIO_LIT. brucei PRMT1 prozyme adopts a chaperone function conserved across kinetoplastids\nC_LI

biochemistry↗