Architecture of the trypanosome RNA editing accessory complex, MRB1

Abstract

Trypanosoma brucei undergoes an essential process of mitochondrial uridine insertion and deletion RNA editing catalyzed by a 20S editosome. The multiprotein mitochondrial RNA-binding complex 1 (MRB1) is emerging as an equally essential component of the trypanosome RNA editing machinery, with additional functions in gRNA and mRNA stabilization. The distinct and overlapping protein compositions of reported MRB1 complexes and diverse MRB1 functions suggest that the complex is composed of subcomplexes with RNA-dependent and independent interactions. To determine the architecture of the MRB1 complex, we performed a comprehensive yeast two-hybrid analysis of 31 reported MRB1 proteins. We also used in vivo analyses of tagged MRB1 components to confirm direct and RNA-mediated interactions. Here, we show that MRB1 contains a core complex comprised of six proteins and maintained by numerous direct interactions. The MRB1 core associates with multiple subcomplexes and proteins through RNA-enhanced or RNA-dependent interactions. These findings provide a framework for interpretation of previous functional studies and suggest that MRB1 is a dynamic complex that coordinates various aspects of mitochondrial gene regulation.

Integrity of the core mitochondrial RNA-binding complex 1 is vital for trypanosome RNA editing

“…Several pentatricopeptide proteins, which are so named because they contain the eponymous RNA-binding motif, have been shown to be membrane associated along with mt ribosomal RNA (Pusnik et al 2007). Among these is the kinetoplast polyadenylation/ uridylation factor 1 (kPAF1), also known as PPR1, which interacts with both MRB1 and mt ribosomes (Aphasizheva et al 2011;Ammerman et al 2012).…”

“…MRB8620 has been shown to interact with the MRB1 core complex and the TbRGG2 subcomplex by yeast two-hybrid analysis and various tandem affinity purification (TAPs) of tagged MRB1 subunits Weng et al 2008;Hernandez et al 2010;Ammerman et al 2011Ammerman et al , 2012Kafková et al 2012). To confirm that MRB8620 is an integral component of the MRB1 complex, we analyzed proteins copurifying with the C-terminally PTP-tagged MRB8620 without nuclease treatment by LC-MS/MS mass spectroscopy.…”

“…The GAP1/2 proteins also represent the only bona fide RNA-binding components of the complex identified to date. RNAi silencing of three other subunits called MRB5390, MRB3010, and MRB11870 (nomenclature by Ammerman et al 2012) also impacts editing without destabilizing gRNAs, with editing stalling at initial stages of pan-editing, presumably guided by the first gRNA:mRNA duplex, in the case of the latter two proteins (Acestor et al 2009;Ammerman et al 2011Ammerman et al , 2013.…”

“…In this report, we describe the functional analysis of MRB8620 (TriTrypDB accession number: Tb927.11.16860; formerly "Tb11.01.8620"), the last remaining MRB1 core subunit as defined by Ammerman et al (2012), by virtue of its consistent presence in various MRB1 core isolations, to be studied this way. This study was performed in the various physiological states of T. brucei in vitro cultures: PS grown in the presence and paucity of glucose and BS that has been rendered kDNA-independent due to bearing a γ-subunit site mutation that allows this condition.…”

The Importance of the 45 S Ribosomal Small Subunit-related Complex for Mitochondrial Translation in Trypanosoma brucei

“…This could be achieved through the components of the SSU* complexes shared with the KPAP polyadenylation complex and/or other parts of the mRNA processing machinery. Several such components have been identified by the proteomics analyses in L. tarentolae and T. brucei, including the PPR29 protein investigated herein and several other PPR (Tb927.11.5500) and non-PPR proteins (Tb927.11.1250, Tb927.11.2530) (17,28,43,46,54). It is noteworthy that this effect is mRNA-specific, as follows from the observation that the LT of the RPS12 mRNA remains unchanged.…”