Analysis of proteins contacting the limiting sequences of RON introns 10 and 11 by crosslinking.

<p>(A) Sequences of transcripts used for UV-crosslinking. These comprised 44 nt and 40 nt from the 3’ ends of intron 10 and 11, respectively, together with a 5’GGG added to enhance transcription. In the natural sequence a GGG motif is found 4 nts and 2 nts 5’ of these intron 10 and 11 sequences, respectively. The start of a shorter intron 10 transcript of 22 nt is shown. (B) Detection by UV-crosslinking and SDS-PAGE of proteins bound to [α-³²P]GTP-labelled transcripts of the 44 nt and 40 nt sequences of introns 10 and 11. Control samples were not irradiated (UV -). Transcripts were incubated in nuclear extracts from HeLa and KATOIII cells. The extent of migration of protein size markers is shown. (C) UV-crosslinking analysis as in (B) to the 44 nt and 40 nt transcripts of introns 10 and 11 after incubation in nuclear extracts from HeLa and HEK293T cells and from HEK293T cells expressing GFP-hnRNP F. (D) Immunoprecipitation of products from UV-crosslinking in HeLa nuclear extract with anti-hnRNP F/H. (E) UV-crosslinking to detect binding of U2AF65 or PTB. Transcripts were labelled by inclusion of [α-³²P]UTP and [α-³²P]GTP during transcription. Transcripts were incubated in HeLa nuclear extract and processed as in panel B. The sequences used comprised the 44 nt and 22 nt portions of intron 10, as in (A). As markers for the two proteins, crosslinking was done also with a portion of <i>Tpm1</i> 5’ of exon 3 that crosslinks readily to PTB at low salt concentrations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077208#B49" target="_blank">49</a>] and, at higher concentrations, to U2AF65 (C. Gooding, personal communication).