TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 59 ends of pan-edited RNAs than at their 39 ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 39 to 59 progression of editing. Detailed analyses of edited RNAs from wildtype and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 39 ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA-RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 39 to 59 progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.

Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3=-to-5= progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. T rypanosome RNA editing entails the precise addition and removal of uridine nucleotides in mitochondrial RNAs. In Trypanosoma brucei, 12 of the 18 mitochondrially encoded mRNAs require editing for maturation prior to their translation. Essential players in this process are mitochondrially encoded 50-to 60-nucleotide (nt)-long guide RNAs (gRNAs), which direct the positions of uridine insertion and deletion through base-pairing interactions. The editing cycle is initiated upon association of a cognate gRNA with preedited mRNA by formation of a short anchor duplex. Editing catalysis is mediated by multiprotein complexes called editosomes or RNA editing core complexes (RECCs), and editing efficiency is achieved through the actions of transiently associating accessory factors (8,16,45,55,56,62,63). Following annealing of gRNA/preedited mRNA, a gRNAdirected endonuclease cleaves the premRNA at the site of gRNA/mRNA mismatch, and U insertion or deletion is catalyzed by terminal uridylyl transferase or U-specific exoribonuclease activities, respectively. The mRNA is then resealed by RNA ligase in preparation for a subsequent editing cycle. The editing cycles continue until gRNA/mRNA base pairing is extended along the entire length of the gRNA. gRNAs are then presumably exchanged, and the process continues, proceeding in a general 3=-to-5= direction along the mRNA. While "minimally edited mRNAs" are edited only in small regions, the majority of mRNAs are edited throughout their lengths and thus are termed panedited. Complete editing of panedited mRNAs requires sequential utilization of dozens of gRNAs.RNA-editing accessory factors are thought to coordinate recruitment of RNAs to the editosome, to direct correct gRNA/ mRNA annealing, and to regulate editing progression by modulating RNA-RNA and RNA-protein interactions. Accessory factors studied to date include RBP16, MRP1/2, and TbRGG2, all of which bind and anneal RNAs, T. brucei RGG1 (TbRGG1), and the RNA helicase REH1 (2,28,33,40,44,50,57,65). TbRGG2 is a component of a multiprotein complex, Mitochondrial RNA Binding Complex 1 (MRB1, also known as GRBC), which contains numerous proteins ...

Background:Entamoeba histolytica has an RNAi-mediated TGS pathway but the epigenetic marks are not known. Results: H3K27Me2 is an inducible repressive histone modification enriched in loci silenced by small RNAs. Conclusion: Transcriptional gene silencing and chromatin modification are coupled to maintain prolonged gene silencing. Significance: This work identifies the first repressive histone mark in Entamoeba and links it to RNAi.

Author contributions: A.E.C. and D.M.S. designed experiments, performed experimental work, analyzed results, prepared figures, and wrote the manuscript. B.F. performed experimental work and provided intellectual contribution. G.P. provided critical cell sorting expertise. M.Y.K. analyzed single-cell RNA sequencing (scRNA-seq) data and revised the manuscript. C.-W.L. analyzed the scRNA-seq data. R.B. generated gene regulatory network models. W.R.D. provided reagents and intellectual contribution. W.C. and Y.-G.C. conceived the study, supervised the project, and revised the manuscript.The scRNA-seq data presented in this article have been submitted to the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE155595) under accession number GSE155595.