Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

Abstract

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3 ′ -to-5 ′ in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNAbinding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3 ′ ends and strain-specific alternative 3 ′ editing within 3 ′ UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.

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

“…As performed in a previous study (Madina et al 2014), we confirmed that cDNAs from mt RNAs were enriched in the GAP1 IP when compared with the mock (Supplemental Fig. 1).…”

“…Taken together, these data indicate that MRB1 core integrity is dependent on subunits such as MRB8620 and MRB11870 , whereas the presence of the GAP1/2 heterotetramer is dispensable for its assembly or stability and even its association with TbRGG2. This finding plus the heterodispersion of the GAP1/2 subcomplex in density gradients (Acestor et al 2009;Hashimi et al 2009;Ammerman et al 2011Ammerman et al , 2012Kafková et al 2012;Aphasizheva et al 2014) and the association of GAP1/2 with REH2 (Madina et al 2014) or TbRGG3 (McAdams et al 2015 suggest that GAP1/2 interactions are not restricted to the MRB1 core and that the heterotetramer represents a functionally distinct entity.…”

“…The shift of GAP1 to more dense fractions upon MRB8620 silencing may reflect its increased accumulation on mRNA as described above. Because GAP1 associates with additional proteins outside the MRB1 complex (Madina et al 2014;McAdams et al 2015), these larger GAP1-containing complexes may also comprise non-MRB1 ribonucleoprotein complexes. Similar shifts of GAP1 sedimentation to larger glycerol gradient fractions has been reported previously in response to depletion of specific MRB1 proteins (Aphasizheva et al 2014).…”

“…This result indicates that edited mRNAs may associate with GAP1/2 subcomplex, given the annealed gRNA: mRNA duplexes as the substrate of RNA editing. The copurification of gRNAs and mRNAs with MRB3010 protein is precedent for this notion (Madina et al 2014).…”

High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing

“…Numerous recent studies have demonstrated that a large, possibly dynamic and heterogeneous complex termed MRB1 (mitochondrial RNA binding complex 1) or RESC (RNA editing substrate binding complex) is also essential for RNA editing in kinetoplastids (Fisk et al 2008;Hashimi et al 2008Hashimi et al , 2009Hashimi et al , 2013Weng et al 2008;Acestor et al 2009;Ammerman et al 2011Ammerman et al , 2012Ammerman et al , 2013Kafkova et al 2012;Aphasizheva et al 2014;Huang et al 2015;Madina et al 2015;Read et al 2016). Indeed, the MRB1 complex likely acts as the platform for RNA editing, since it contains readily detectable mRNA and gRNA, while purified RECC lacks associated RNA (Weng et al 2008;Aphasizheva et al 2014;Madina et al 2014Madina et al , 2015. MRB1 appears to be composed of interacting subcomplexes with distinct roles in the editing process, and it transiently associates with other proteins that impact mitochondrial RNA editing and processing (Hashimi et al 2008(Hashimi et al , 2009Weng et al 2008;Ammerman et al 2011Ammerman et al , 2012Kafkova et al 2012;Aphasizheva et al 2014;Madina et al 2014Madina et al , 2015Read et al 2016).…”