MRB10130 is a RESC assembly factor that promotes kinetoplastid RNA editing initiation and progression

Abstract

Uridine insertion deletion editing in kinetoplastid protozoa requires a complex machinery, a primary component of which is the RNA editing substrate binding complex (RESC). RESC contains two modules termed GRBC (guide RNA binding complex) and REMC (RNA editing mediator complex), although how interactions between these modules and their mRNA and gRNA binding partners are controlled is not well understood. Here, we demonstrate that the ARM/HEAT repeat containing RESC protein, MRB10130, controls REMC association with mRNA-and gRNA-loaded GRBC. High-throughput sequencing analyses show that MRB10130 functions in both initiation and 3 ′ ′ ′ ′ ′ to 5 ′ ′ ′ ′ ′ progression of editing through gRNA-defined domains. Editing intermediates that accumulate upon MRB10130 depletion significantly intersect those in cells depleted of another RESC organizer, MRB7260, but are distinct from those in cells depleted of specific REMC proteins. We present a model in which MRB10130 coordinates numerous protein-protein and protein-RNA interactions during editing progression.

Trypanosome RNAEditing Substrate Binding Complex integrity and function depends on the upstream action of RESC10

“…The levels of target proteins were normalized by western blot using either specific antibodies against the target or antibodies recognizing the tag, as indicated on each figure. Co-purifying proteins were then detected by western blotting with specific antibodies against RESC6 ( 23 ), RESC13 ( 40 ), RESC11A ( 12 ), RESC12A ( 43 ), RESC8 ( 29 ), RESC14 ( 25 ), RESC10 (this study) and RESC2 ( 33 ).…”

“…PF T. brucei RESC6-PTP and RESC13-HTM cells harboring the RESC10 RNAi construct were grown in presence or absence of 4 μg/ml of doxycycline for 3 days. Cells (1 × 10 10 ) were collected and mitochondria enriched as described above, and RIP was performed as described previously ( 25 , 29 ). Briefly, RESC6-PTP and RESC13-HTM cells were immunoprecipitated with IgG fast flow (GE Healthcare) and Myc (ICL) beads, respectively.…”

“…The supernatant was removed, and RNA was extracted with phenol/chloroform. RNA was DNase-treated (Ambion DNA-free DNase Kit) and 500 ng of RNA converted to cDNA with T. brucei established gene specific primers were used, with normalization to 18S rRNA gene ( 25 , 29 ) using the iScript cDNA synthesis kit (BioRad). cDNA was amplified by using SsoAdvanced PreAmp Supermix (BioRad) and then used for qRT-PCR.…”

“…18S rRNA was used for normalization. Fold change was determined using ΔΔCt method as described previously ( 25 , 29 ).…”

“…The paralogous RNA binding proteins RESC12A and RESC12 also bind RESC13, and they function in editing initiation, likely facilitating recruitment of other RESC proteins, as well as to constrain the region of active editing ( 12 , 28 ). Recent evidence indicates that the ARM/HEAT repeat containing RESC8, previously denoted as a REMC protein ( 19 ), instead functions as a RESC organizer in the recruitment and sequential exchange of RESC factors and promotion of protein-protein and protein–RNA rearrangements during editing ( 29 ). Finally, RESC14, designated as neither a REMC nor GRBC component, plays a role in modulating RNA–protein interactions within RESC, and promotes editing progression through impacts on gRNA utilization.…”