Fig. 2.

TcUBP pre-mRNA is processed by trans-splicing. (A) Scheme of the pR-TcUBP DNA construct (dicistron with TcUBP1 CDS fused to GFP) made in pRIBOTEX vector. (B) Northern blot using RNA obtained from transfected parasites and hybridized with a GFP probe (Left); ∗ denotes the position of a band of ≈5 kb detectable when the film was overexposed. RT-PCR was performed by using total RNA extracted from pR-TcUBP transfected parasites. cDNA was synthesized by using a COOH-GFP primer, and the PCR was performed with TcSL and GFP as primers (see SI Table 3). The PCR products were separated in an agarose gel and visualized by ethidium bromide staining (Right). (C) Schematics of endogenous and recombinant TcUBP1 monocistronic mRNAs, showing that both share the same 5′ UTR. (D) Western blot was performed by using total protein extracts from transfected parasites (pR and pR-TcUBP) and probed with polyclonal rabbit anti-RNA-recognition motif sera (1/1,000 dilution). (E) Fluorescence microscopy of a T. cruzi insect-stage parasite transfected with the pR-TcUBP construct shown in A. 3′ SS, acceptor splice site; pPy, polypyrimidine tract; N, nuclear DNA; K, kinetoplast DNA. The molecular weight markers are shown on the right.