Native snRNP protein (total protein) preparations and in vitro reconstitution of

The successful synthesis of these chimeric U2 snRNA molecules allowed their investigation in the two-step U2 snRNP reconstitution/complementation assay.

4.1.2 Native snRNP protein (total protein) preparations and in vitro reconstitution of core U2 snRNPs

Reconstitution of U2 snRNP is achieved by a two-step procedure (Fig. 4-3). In the first step, U2 snRNA is incubated with native, RNA-free snRNP proteins (denoted total proteins or TPs) to form 12S core U2 snRNPs. Since TPs lack the 17S U2

snRNP-Figure 4-2 . Ligations performed according to Moore and Sharp ligation procedure.

3' fragment of U2 snRNA (a U2 transcript from nucleotides 25 to 192) was ligated with a series of U2 5' oligos which are shown in lanes 2-5. Lane 2: Ligation with

&#m oligo containing no modifications; lane 3: ligation with &m oligo containing only pseudouridines; lane 4: ligation with &# oligo containing only 2'-O-methyl groups; lane 5: ligation with #m oligo containing both of the modifications (see Fig. 4-1A). Lane 6 shows the input 3' fragment for the ligation reaction. Lane 1 shows the ³²P-pCp-labelled native U2 snRNA. The difference between native U2 snRNA (189 nts) and ligated ones comes from the extra three G nts after 189th in ligated U2 snRNAs. The ligation mixtures were loaded directly onto 9.6 % polyacrylamide-8.3 M urea gel and gel-purified.

specific proteins, the reconstitution of splicing active forms of U2 snRNP requires an additional incubation step (the second step in the two step reconstitution/

complementation system) with nuclear extract. Therefore, in the second step, these 12S U2 snRNPs were incubated with U2-depleted nuclear extract to produce fully active 17S U2 snRNPs. By adding radiolabelled pre-mRNA, the spliceosomal complex formation and splicing were analyzed. In order to reconstitute core U2 snRNPs, it was thus necessary first to prepare the total proteins.

Figure 4-3 . The two-step reconstitution/complementation system. In the first step, the U2 snRNPs are depleted from the nuclear extract at high salt concentration (0.8 M). The U2-specific proteins (SF3a and SF3b protein complexes) are retained in the nuclear extract due to high salt. In the second step, the core U2 snRNPs were reconstituted in vitro and incubated with the U2-depleted nuclear extract which contains the U2-specific proteins at low salt concentration (0.1 M). Finally, mature 17S U2 snRNPs are formed and used in splicing assays (see Materials and Methods).

4.1.2.1 Native snRNP protein (total protein) preparations

Total proteins were prepared from a mixture of U1, U2, U5 and U4/U6 snRNPs which had been isolated according to Kastner et al. (1998). This mixture contained predominantly U1 snRNP and traces of U2, U4/U5.U6 snRNP (see lane 3, in Fig. 4-4 for the RNA composition). In contrast to most of the specific proteins, the core proteins are stably associated with the snRNAs. Thus, dissociation of the snRNP core particle without denaturation of the core proteins requires particular conditions. The protein-RNA interactions within the snRNP particle are first weakened by chelation of divalent cations with EDTA. The proteins are subsequently separated from the RNA by ion-exchange chromatography over the polycationic resin DE53 (Sumpter et al., 1992) forming the total proteins. The U snRNAs were quantitatively removed during the TP preparation as shown in lane 2 in Fig. 4-4. The native snRNP protein preparation used for U2 snRNP reconstitution is thus essentially RNA-free.

The composition of these native snRNP preparations is shown in Fig. 4-5. They contain predominantly the snRNP core proteins (Sm proteins), B, B', D1, D2, D3, E, F and G.

Figure 4-4. Analysis of the RNA content of native snRNP protein (TP) preparation. RNA was isolated by Proteinase K digestion and phenol/chloroform extraction of 5 #g TPs (lane 2) and 5 #g U1-U6 snRNPs (lane 3). Lane 1 shows 0.2 #g native U2 snRNA for control. snRNAs were fractionated on a 9.6 % polyacrylamide-8.3 M urea gel and visualized by staining with ethidium bromide.

Compared to the Sm proteins, the particle specific proteins, in particular, U2 A' and U2 B'' proteins, are underrepresented. As observed by others (Raker, 2000), the U1 70K protein was lost during the TP preparations.

Figure 4-5. Protein composition of native snRNP protein (TP) preparations. TPs used for U2 snRNP reconstitution were prepared as described in Materials and Methods, were fractionated on a 13 % SDS-polyacrylamide - high TEMED gel and stained with Coomassie. 23 #g of the TP preparation used in in vitro reconstitutions in these experiments is shown in lane 1. 32 #g U snRNPs are shown in lane 6 and M shows marker (NOVEX).

4.1.2.2 In vitro reconstitution of core U2 snRNP

After the preparation of total proteins, the first step of the two-step reconstitution/

complementation system was carried out by incubating the HeLa, in vitro transcribed or chimaeric U2 snRNAs with TPs to form core 12S U2 snRNPs. The formation of in vitro reconstituted core U2 snRNP particles includes binding of Sm proteins to Sm binding site. As shown in Fig. 4-6, native and in vitro transcribed U2 snRNA can form core U2 snRNPs successfully.

Previous studies (Raker et al., 1999) showed that the minimal requirement for the Sm core RNP was a nonameric Sm site RNA oligonucleotide. This shows that core U2 snRNPs will form on the Sm binding site of U2 snRNAs irrespective of the presence or absence of modifications within the first 24 nucleotides since the chimaeric U2 snRNAs have their Sm binding site.

Figure 4-6. Native gel analysis of core U2 snRNP formation. Core U2 snRNPs were reconstituted with the ³²P-pCp labelled HeLa U2 snRNA or in vitro transcribed U2 snRNA. Reconstitutions were analyzed on 6 % polyacrylamide non-denaturing gel and visualized by autoradiography. Lane 2 and 3 show core U2 snRNP reconstituted with the HeLa U2 snRNA and in vitro transcribed U2 snRNA, respectively. HeLa U2 snRNA is shown in lane 1 for control.

The 12S core U2 snRNPs were needed to be incubated with the U2-depleted nuclear extract to form fully active 17S U2 snRNP.