Genome Sequences of Five Indian Canine Rabies Virus Isolates Obtained Using Oxford Nanopore Technologies Sequencing.

We report five canine rabies virus genome sequences from India that were obtained from brain samples using Oxford Nanopore Technologies sequencing. The sequences will facilitate understanding of the evolution and transmission of rabies.

The role of nuclear organization in trans-splicing based expression of heat shock protein 90 in Giardia lamblia.

Hsp90 gene of G. lamblia has a split nature comprising two ORFs separated by 777 kb on chromosome 5. The ORFs of the split gene on chromosome 5 undergo transcription to generate independent pre-mRNAs that join by a unique trans-splicing reaction that remains partially understood. The canonical cis-acting nucleotide elements such as 5'SS-GU, 3'SS-AG, polypyrimidine tract and branch point adenine are present in the independent pre-mRNAs and therefore trans-splicing of Hsp90 must be assisted by spliceosomes in vivo. Using an approach of RNA-protein pull down, we show that an RNA helicase selectively interacts with HspN pre-mRNA. Our experiments involving high resolution chromosome conformation capture technology as well as DNA FISH show that the trans-spliced genes of Giardia are in three-dimensional spatial proximity in the nucleus. Altogether our study provides a glimpse into the in vivo mechanisms involving protein factors as well as chromatin structure to facilitate the unique inter-molecular post-transcriptional stitching of split genes in G. lamblia.

Giardia lamblia Hsp90 pre-mRNAs 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 ORFs localized 777 kb apart. The pre-mRNAs transcribed from these ORFs 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-mRNAs have the capability to self-splice in the absence of nuclear proteins. Inhibition of the trans-splicing reaction using a ssDNA 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.