The 3′-untranslated region of cytochrome oxidase II mRNA functions in RNA editing of African trypanosomes exclusively as a cis guide RNA

Abstract

RNA editing in trypanosomes is a post-transcriptional process responsible for correcting the coding sequences of many mitochondrial mRNAs. Uridines are specifically added or deleted from mRNA by an enzymatic cascade in which a pre-edited mRNA is specifically cleaved, uridines are added or removed, and the corrected mRNA is ligated. The process is directed by RNA molecules, termed guide RNAs (gRNA). The ability of this class of small, noncoding RNA to function in RNA editing is essential for these organisms. Typically, gRNAs are transcribed independent of the their cognate mRNA and anneal to form a binary RNA complex . An exception for this process may be cytochrome oxidase subunit II (COII) mRNA since a gene encoding a trans acting gRNA has not been identified. Using an in vitro editing assay we find that the 3 UTR of COII, indeed, functions as a guide for both the site and number of uridines added to the coding region of the COII mRNA. We further show that the guiding sequence within the COII 3 UTR can only function in COII editing when contiguous with the editing substrate, indicating that the 3 UTR of COII lacks sequence or structure information necessary to function as a trans-acting gRNA. While other RNAs have been shown to "guide" RNA processing reactions, our discovery that the COII 3 UTR directs editing of its cognate mRNA in cis, is a unique function for a 3 UTR. The findings described here have led us to propose a new model for the evolution of gRNAs in kinetoplastids.

TbRGG1, an essential protein involved in kinetoplastid RNA metabolism that is associated with a novel multiprotein complex

“…However, the reduction of edited co2 mRNA in the TbRGG1 knockdowns is quite telling, since its gRNA is located within its 39-UTR, thus not requiring the independent trans-acting gRNA molecules (Kim et al 1994). TbRGG1 binding to gRNAs, which are heterogeneous in sequence, would most likely involve conserved features such as the 39 oligo(U) tail, which are absent in the co2 cis-gRNA, as implied by its incapacity to act in trans (Golden and Hajduk 2005). Thus, based on the observation that co2-edited RNAs are affected by TbRGG1 depletion, while gRNA levels are not, we conclude that the protein does not act through these small RNAs.…”

Kinetoplastid guide RNA biogenesis is dependent on subunits of the mitochondrial RNA binding complex 1 and mitochondrial RNA polymerase

“…Significantly, pre-edited and edited cox2 mRNAs were also not affected by RNAimediated elimination of either GAP. This transcript is unique in that its single gRNA is contained in its 39-untranslated region, acting as a guide for insertion editing in cis (Golden and Hajduk 2005). The assay also shows that dsRNAs specifically target only the intended GAP RNA (Fig.…”

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

“…From the mRNAs that require only modest U insertions, called minimally edited, only cytochrome c reductase subunit b (CYB) shows a ∼30% decrease and ∼20% increase in the edited and pre-edited species, respectively. The persistence of CO2 editing upon ablation of MRB8620 is notable, because it uniquely does not require a trans-acting gRNA for its editing, but uses a gRNA-like element within its 3 ′ -UTR (Golden and Hajduk 2005). The pan-edited mRNAs, which require U insertion/deletion throughout the transcript, are most affected in the MRB8620 knockdowns (Fig.…”

“…If the ES is an insertion site, the RECC terminal U transferase (KRET2) appends the 5 ′ fragment with the titular nucleotide (Ernst et al 2003). The mRNA encoding cytochrome c oxidase (cox) 2 is cut by the third RECC endonuclease that recognizes this unique substrate, which contains a gRNA-like element in its 3 ′ UTR that guides the addition of 4 U's within the ORF by KRET2 (Golden and Hajduk 2005). After the appropriate editing event is finished at the ES, an RNA ligase reseals the two mRNA fragments Verner et al 2015).…”