Trypanosome RNA Editing Mediator Complex proteins have distinct functions in gRNA utilization

Abstract

Uridine insertion/deletion RNA editing is an essential process in kinetoplastid parasites whereby mitochondrial mRNAs are modified through the specific insertion and deletion of uridines to generate functional open reading frames, many of which encode components of the mitochondrial respiratory chain. The roles of numerous non-enzymatic editing factors have remained opaque given the limitations of conventional methods to interrogate the order and mechanism by which editing progresses and thus roles of individual proteins. Here, we examined whole populations of partially edited sequences using high throughput sequencing and a novel bioinformatic platform, the Trypanosome RNA Editing Alignment Tool (TREAT), to elucidate the roles of three proteins in the RNA Editing Mediator Complex (REMC). We determined that the factors examined function in the progression of editing through a gRNA; however, they have distinct roles and REMC is likely heterogeneous in composition. We provide the first evidence that editing can proceed through numerous paths within a single gRNA and that non-linear modifications are essential, generating commonly observed junction regions. Our data support a model in which RNA editing is executed via multiple paths that necessitate successive re-modification of junction regions facilitated, in part, by the REMC variant containing TbRGG2 and MRB8180.

MRB7260 is essential for productive protein–RNA interactions within the RNA editing substrate binding complex during trypanosome RNA editing

“…REMC is currently envisioned as comprised of TbRGG2, MRB8180, MRB800, MRB1860, and the redundant paralogs MRB8170/4160. It associates with GRBC in an RNA-enhanced manner, and appears to be more heterogeneous than GRBC Kafková et al 2012;Aphasizheva et al 2014;Simpson et al 2017). MRB10130, which contains domains that resemble Armadillo (ARM) or HEAT repeats, may play a role in organizing macromolecular interactions within RESC.…”

“…GAP1/2 are the only proteins identified to date required for gRNA stabilization (Weng et al 2008;Hashimi et al 2009), while the remaining GRBC components appear to impact the initiation of RNA editing as indicated by an accumulation of pre-edited mRNA in cells depleted of these proteins (Weng et al 2008;Hashimi et al 2009;Ammerman et al 2011Ammerman et al , 2012Ammerman et al , 2013Aphasizheva et al 2014;Huang et al 2015). We recently used high-throughput sequencing analysis of partially edited transcripts in cells depleted of specific RESC components in combination with a novel bioinformatic platform, termed TREAT (trypanosome RNA editing alignment tool), to address RESC protein function (Simpson et al 2017). We demonstrated that mRNAs from cells depleted of GAP1 display both a defect in editing initiation as well as pausing in the 3 ′ to 5 ′ progression of editing consistent with a defect in gRNA exchange (Simpson et al 2017).…”

“…We recently used high-throughput sequencing analysis of partially edited transcripts in cells depleted of specific RESC components in combination with a novel bioinformatic platform, termed TREAT (trypanosome RNA editing alignment tool), to address RESC protein function (Simpson et al 2017). We demonstrated that mRNAs from cells depleted of GAP1 display both a defect in editing initiation as well as pausing in the 3 ′ to 5 ′ progression of editing consistent with a defect in gRNA exchange (Simpson et al 2017). However, in this case, pausing at the ends of gRNA-defined blocks is potentially due to destabilization of the entire gRNA population rather than an active role for GAP1/2 in the process of gRNA exchange.…”

“…With regard to REMC, we demonstrated that TbRGG2 is critical for the 3 ′ to 5 ′ progression of editing and functions as an RNA binding, annealing, and unwinding protein (Fisk et al 2008;Ammerman et al 2010;Foda et al 2012). More recently, our high-throughput sequencing analysis of partially edited transcripts demonstrated that TbRGG2 and MRB8180 are involved in the utilization of gRNAs (Simpson et al 2017). At least one function of these proteins is to promote the commonly observed nonlinear path of editing that results in junctions containing incorrectly edited sequence at the 5 ′ leading edge of editing, likely through modulation of gRNA-mRNA pairing (Simpson et al 2017).…”

“…More recently, our high-throughput sequencing analysis of partially edited transcripts demonstrated that TbRGG2 and MRB8180 are involved in the utilization of gRNAs (Simpson et al 2017). At least one function of these proteins is to promote the commonly observed nonlinear path of editing that results in junctions containing incorrectly edited sequence at the 5 ′ leading edge of editing, likely through modulation of gRNA-mRNA pairing (Simpson et al 2017). Studies from our laboratory and others addressing the function of another REMC protein, MRB8170, indicate that this protein functions differently than TbRGG2 and MRB8180.…”