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. 2010 Feb 16;38(10):e114. doi: 10.1093/nar/gkq065

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© The Author(s) 2010. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — (A) Schematic presentation of a trypanosome polycistronic pre-mRNA. The significant sequence elements essential for trans-splicing are indicated. (B) Pre-mRNA substrates used in this study. Schematic representation of the pre-mRNA substrates highlighting the significant sequence elements: polyadenylation site (polyA site), polypyrimidine tract (PPT) and AG splice site. The lengths of the PIR and TIR UTRs as well as the Luciferase ORF are indicated. Underneath each pre-mRNA, the respective probe, in vitro trans-spliced product and endogenous mRNA are shown, along with the expected size of the protected fragments after RNase protection. (C) RNase protection assay to monitor the expression of the TIR and PIR constructs. (a) Thirty micrograms of total RNA was hybridized with labeled antisense probe, complementary to the trans-spliced product, as described in ‘Materials and Methods’ section. Protected RNA products were separated on a 6% sequencing gel. M- DNA marker, labeled pBR322 DNA MspI digest. The size of the marker is indicated on the left. Lane contents are as follows: 1, RNA from parental strain; 2, RNA from transgenic parasites expressing the pNS21-PIR construct; 3, RNA from transgenic parasites expressing the pNS21-TIR construct. The scheme on the right hand side of the gel indicates the structure of the probe and the protected fragments. (b) Relative expression of the tubulin-luciferase or the EP-luciferase to the endogenous tubulin and EP transcripts.