Giardia lamblia Hsp90 pre‐ mRNA s undergo self‐splicing to generate mature RNA in an in vitro trans‐splicing reaction

We have previously shown that the Heat Shock Protein 90 (Hsp90) gene in G. lamblia is expressed from two ORF s localized 777 kb apart. The pre‐ mRNA s transcribed from these ORF s are stitched by a trans‐splicing mechanism. Here, we provide mechanistic details of this process by reconstituting the reaction using in vitro synthesized pre‐ mRNA substrates. Using RT ‐ PCR , northern blot and nanostring technology, we demonstrate that the in vitro synthesized pre‐ mRNA s have the capability to self‐splice in the absence of nuclear proteins. Inhibition of the trans‐splicing reaction using a ss DNA oligo corresponding to a 26‐nucleotide complementary sequence confirmed their role in juxtapositioning the pre‐ mRNA substrates during the self‐splicing reaction. Our study provides the first example of a self catalysed, trans‐splicing reaction in eukaryotes.