Identification of additional proteins stabilised by the GAR ESE

The binding pattern of hTra2-E to intron/exon 5 RNA targets indicated that besides SF2/ASF and SRp40 additional proteins might contribute to the GAR enhancer function and also revealed enhanced binding of hTra2-E in the presence of the PPT/BPS extension. Therefore, it was aimed (i) to identify additional proteins binding to the RNA dependent on the GAR ESE sequence and (ii) to analyse whether proteins other than U2AF⁶⁵ and SF1/mBBP bind to the PPT/BPS sequence in extended RNA targets, which might further stabilise hTra2-E binding to the RNA and thereby promote exon recognition. To this end, proteins isolated by RNA affinity chromatography as performed before were separated by two-dimensional gel electrophoresis to detect differences hidden till this point in the vast numbers of proteins bound to the respective RNA sequences (Fig. III-16 and Fig. III-17A).

To identify proteins binding GAR ESE-dependent to the RNA, proteins isolated by RNA affinity chromatography using short RNA targets containing the minimal PPT upstream and the GAR ESE downstream of the 3’ ss A5 (Fig. III-18A, A5 GAR ESE) were compared to the proteins isolated using RNA targets of identical length carrying

Fig. III-18: Analyses of RNA affinity purified proteins by 2D-gel electrophoresis.

Triplicates of RNA affinity chromatography reactions including phosphatase inhibitors were performed for RNA targets excluding (A) or including the BPS upstream of 3’ ss A5 (B) as depicted in Fig. III-14A.

Entire protein preparations were passively loaded onto IPG strips pH 3-10 (Amersham) and after isoelectric focusing (Ettan IPGphor III) mounted on 10% SDS-polyacrylamide minigels for separation in the second dimension. Proteins were visualised by sensitive coomassie blue staining using a protocol appropriate for subsequent mass spectrometry analyses (299). As control for RNA specific protein binding affinity chromatography reactions were also performed in the absence of RNA (see Appendix, Fig. VII-2).

A sequence (short RNA targets). The sketch illustrates SF2/ASF and SRp40 binding to the GAR ESE and the questioned enhancer-mediated binding of potential splicing regulatory proteins. Protein spots derived from the RNA target containing the parental GAR ESE (A5 GAR ESE, reference) were compared to the protein spots resulting from RNA affinity chromatography of the respective RNA targets containing either mutations in the SF2/ASF binding sites and in the SRp40 binding site (A5 ESE-) or only in two SF2/ASF binding sites (A5 2xSF2-).

(B) Analysis of proteins binding GAR ESE-mediated to RNA targets carrying an extended PPT (extended RNA targets). Protein spots identified after RNA affinity chromatography of RNA targets additionally containing a PPT extension and the most proximal BPS A5 (BPS A5 GAR ESE, reference) were first compared to protein spots derived for the corresponding RNA target lacking the 5’-extension (Fig. III-18A, A5 GAR ESE) yielding 41 protein isoforms binding to the RNA target dependent on the 5’-extension. To determine, which of these proteins bind GAR ESE-mediated to the 5’-extension, protein spots were in a second analysis compared to their respective positional counterparts in 2D-gels derived from RNA affinity

mutations in the GAR ESE (Fig. III-18A, A5 ESE-). To further refine the subgroup of proteins contributing to exon recognition, the simultaneous mutation of two SF2/ASF binding sites of the GAR ESE was used as control to eliminate proteins which might be involved in 3’ ss selection, but which still allow low levels of exon recognition and therefore interfere with the detection of proteins essentially mediating exon recognition (Fig. III-18A, A5 2xSF2-). Proteins were first separated according to their isoelectric point (pI) using an immobilised pH gradient and in the second dimension according to their size (Fig. III-18A). Since calculated pI-values (www.phosphosite.org) for representative splicing regulatory proteins were found to extend into the alkaline region, a broad pH-gradient ranging from pH 3-10 was chosen for isoelectric focusing in the first dimension. Nevertheless, binding of vast numbers of protein isoforms with alkaline pI-values to the RNA targets impaired their differentiation into individual proteins spots resulting in an only suboptimal separation resolution in the alkaline area.

After dividing the protein population binding to the RNA target containing the minimal PPT into 468 protein isoforms represented by individual protein spots, comparison with protein isoforms identified to bind to the RNA target carrying a triple mutation in the GAR ESE (ESE-) revealed 39 proteins lacking a matching protein spot after inactivation of the GAR ESE (Fig. III-18A). Comparison of these proteins with 35 protein isoforms, which remained unmatched after mutation of two SF2/ASF binding sites (2xSF2-), showed that only 17 of the 39 proteins absent after inactivation of the GAR ESE were specific for this mutation (Tab. III-1). These proteins were considered as candidate isoforms binding GAR ESE-mediated to RNA targets carrying the minimal PPT (A5) or to the BPS of extended RNA targets (BPS A5).

A

Fig. III-19: Depiction of protein isoforms hypothesised to regulate exon recognition bound to BPS-lacking and BPS–containing RNA targets isolated for identification by mass spectrometry.

Protein spots reduced in volume or absent in 2D-SDS-polyacrylamide gels derived from mutant enhancer sequences (cf. Fig. III-18) were isolated from reference gels derived from parental GAR ESE-containing RNA targets in the absence [(A), A5 GAR ESE] or presence of the most proximal BPS [(B), BPS A5 GAR ESE] and analysed by ESI-Quad-TOF mass spectrometry. Schemes of the respective RNA targets are depicted on the right. Spots isolated for MS analyses are encircled in green. Spot numbers (red) were assigned by ImageMaster 2D Platinum 6.0 software (GE Healthcare) during gel analyses.

proteins contributing to GAR ESE-mediated exon recognition. Besides the complete loss of protein binding also reduced binding of regulatory proteins might decrease exon recognition. After inactivation of the GAR ESE using the triple mutation (ESE-), 22 protein isoforms binding at least 2.5-fold less to the RNA target were found specific for the inactivation of the GAR enhancer. Proteins characterised by specifically abrogated or diminished binding to the RNA after inactivating the GAR ESE (ESE-) were isolated (Fig. III-18C, MS, and Fig. III-19A) and are currently subjected to mass spectrometry analyses. The first proteins identified from that screen are Nucleolin, GTF II, hnRNP Q and hnRNP H (Fig. III-19A).

In a second experiment, proteins binding to the set of extended RNA targets were analysed by 2D-gel electrophoresis to identify proteins binding GAR ESE-dependent to the 5’-extension. To this end, the two dimensional separation of proteins bound to the BPS-containing RNA target in the presence of the GAR ESE (Fig. III-18B, BPS A5 GAR ESE) was compared to proteins bound to the RNA target lacking the BPS (Fig. III-18A, A5 GAR ESE). In this first comparison, 215 of 544 protein spots identified for the BPS-containing GAR ESE RNA target were found to bind specifically to the extended RNA target (Fig. III-18B, BPS A5 GAR ESE). These proteins were further classified by application of a threshold of 0.04 for the absolute protein volume of the protein spot.

The threshold was defined as the lowest absolute protein volume allowing visual identification of the protein spots in coomassie-stained gels thereby enabling their isolation and subsequent sequencing. Application of the threshold narrowed the number of identifiable protein spots down to 41 of the 215 protein spots initially found to bind BPS-mediated to the RNA target. Comparison of the 41 protein spots with their positional counterparts in case the GAR ESE had been mutated yielded 32 putative GAR ESE-dependent protein isoforms binding BPS-mediated to the RNA target. From this group 9 proteins, which were absent exclusively after inactivation of the GAR ESE, might contribute to exon inclusion. The remaining 23 protein spots, which were absent or reduced after mutation of the SF2/ASF binding sites alone or after inactivation of the GAR ESE as well as mutation of the SF2/ASF binding sites, might play only a minor role in exon inclusion, but are likely to contribute to 3’ ss selection. The most promising candidates showing differences between intronic sequences followed by wild-type GAR ESE or mutated enhancer sequences (Fig. III-18D, MS and Fig. III-19B) were isolated and are currently analysed by mass spectrometry (Dr. W. Bouschen, BMFZ, HHUD).

C.1.3.5 U2AF heterodimer binding to the 3’ ss (“complex formation”) requires a