1. Functional Recognition of the 5′ Splice Site by U4/U6
Functional Recognition of the 5′ Splice Site by U4/U6.U5 tri-snRNP Defines a Novel ATP-Dependent Step in Early Spliceosome Assembly 
Patricia A. Maroney, Charles M. Romfo, Timothy W. Nilsen 
Molecular Cell 
Volume 6, Issue 2, Pages (August 2000)
DOI: /S (00)

2. Figure 1 Trans-Splicing as a Reporter for cis 5′ Splice Site Function
(A) The basis of the competition assay between cis- and trans-splicing is shown schematically. The presence of a functional 5′ donor site in cis prevents use of the splice acceptor as a site of SL addition; inactivation of the donor site (represented by the X over the 5′ splice site) permits trans-splicing. For the pre-mRNA splicing substrates used, U2 snRNP recruitment is 5′ splice site independent (see Results).
(B) In vitro splicing of the indicated 3′ end-labeled substrates was carried out as described in the Experimental Procedures, and identities of splicing intermediates and products are indicated schematically. In lane 3, the 5′ splice site was mutant in nucleotides +1 to +6 (indicated by the X). The RNA migrating between the substrate and trans- spliced product in lane 3 is a cis-spliced product resulting from the inefficient use of a cryptic 5′ splice site activated by the mutation.
(C) Splicing in the presence of no oligonucleotide, lane 1; a 2′Ome oligonucleotide complementary to nucleotides 1–14 of the 5′ end of U1 snRNA, lane 2; a 2′Ome oligonucleotide complementary to nucleotides −4 to +10 of the 5′ splice site region of the substrate, lane 3. Lane 4 is a reaction containing the same substrate as B, lane 3. Substrate designations are as in (B).
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2
Differential Activation of trans-Splicing by Specific Mutations in the cis-5′ Splice Site
(A) In vitro splicing of 3′ end-labeled substrates containing either wild-type (lane 1) or two nucleotide 5′ splice site mutations as indicated (lanes 2 through 5) were carried out as described in the Experimental Procedures; the substrate in lane 6 was mutant in nucleotides +1 to +6. Cis- and trans-spliced products are indicated. The potential for base pairing between the wild-type 5′ splice site and the 5′ end of Ascaris U1 snRNA (Shambaugh et al. 1994) is shown on the right.
(B) The same substrates used in (A) were spliced in vitro at either 30°C or 37°C. The RNA resulting from use of the cryptic 5′ splice site described in the legend to Figure 1 is evident in lanes 3, 4, 5, and 11.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3
Sequence-Specific Interaction of a High Molecular Weight Protein with the 5′ Splice Site
(A) Wild-type cis- splicing substrate (Figure 1, lane 1) was labeled with a single radioactive phosphate between the invariant GU of the 5′ splice site as described in the Experimental Procedures. This RNA was then incubated under splicing conditions in the presence of a 2′Ome oligonucleotide complementary to the branch point recognition sequence of U2 snRNA (see the Experimental Procedures). Following incubation, reactions were either mock irradiated (lane 1) or irradiated (lane 2) with 254 nm UV light. Following digestion of the RNA with ribonuclease, labeled proteins were fractionated on 10% SDS polyacrylamide gels and visualized by autoradiography as described in Experimental Procedures. The positions of protein molecular weight standards are indicated.
(B) Wild-type or 5′ splice site mutant RNAs were site specifically labeled as in (A). Incubation, UV irradiation, and labeled protein visualization were exactly as in (A) lane 2.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
Prp8 Interacts with the 5′ Splice Site in the Absence of U2 snRNP Binding to the Branch Point Region
(A) Wild-type or 3′ splice site mutant (AG→UC) RNAs were site specifically labeled at the 5′ splice site as in Figure 3. The two RNAs were then incubated as described in the legend to Figure 3 in either HeLa cell or Ascaris extract. Cross-linking and processing of labeled proteins was exactly as described in the legend to Figure 3.
(B) Cross-linking reactions of 3′ splice site mutant RNAs identical to those in A (lanes 2 and 4) were performed in HeLa cell or Ascaris extracts. Following ribonuclease digestion, aliquots were analyzed directly on SDS gels (lanes 1 and 4), after binding to protein A agarose (lanes 2 and 5), or after binding to anti-Prp8 polyclonal antisera prebound to protein A agarose (lanes 3 and 6). Denaturing immunoprecipitations were as described (Luo et al. 1999).
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5
Inactivation of Either U6 or U4 snRNPs Prevents Association of Prp8 with the 5′ Splice Site
Cross-linking analysis of 5′ splice site labeled pre-mRNAs bearing a 3′ splice site mutation were carried out in HeLa cell extract as described in the legend to Figure 4. The extract was preincubated with the indicated 2′Ome oligonucleotides (lanes 1–5) or DNA oligonucleotides and RNase H (lanes 6–8) as described in the Experimental Procedures. The regions of complementarity between the 2′Ome oligonucleotides and human U6 or U4 snRNAs are depicted schematically. The U6c and U6a oligonucleotides partially overlap, as indicated by the magenta coloring.
Molecular Cell 2000 6, DOI: ( /S (00) )

7. Figure 6
The Prp8/5′ Splice Site Interaction Requires ATP and Incubation at 30°C
Cross-linking reactions using site-specifically labeled pre-mRNA with a mutant 3′ splice site (AG→UC) were carried out in untreated HeLa cell extract (lane 1); extract that had been preincubated to deplete ATP as described in the Experimental Procedures (lane 2); or in depleted extract that was supplemented with ATP (lane 3). In lane 4, the sample contained ATP but was kept on ice. Lane 5 was identical to lane 1. Incubation conditions are described in the Experimental Procedures.
Molecular Cell 2000 6, DOI: ( /S (00) )

8. Figure 7
U4/U6.U5 tri-snRNP-Dependent Protection of the 5′ Splice Site Region
Pre-mRNA (3′ SS AG→UC), with a specific label at the 5′ splice site, was incubated in HeLa cell extract under splicing conditions for 10 min at 30°C. Following incubation, reactions were placed on ice and digested with microccocal nuclease prior to deproteinization (see the Experimental Procedures). Nuclease resistant RNA fragments were fractionated on denaturing gels and visualized by autoradiography. In lane 1, incubation was terminated by nuclease digestion immediately following addition of labeled RNA. In lanes 2 through 6, the extract was preincubated with the indicated 2′Ome oligonucleotides. In lanes 7 through 9, incubations were exactly as described for lanes 1 through 3 in Figure 6. Lane 10 was treated the same as lane 1, and lane 12 was treated the same as lane 2; lane 11 was incubated on ice. The boundaries of protected fragment sets A and B (indicated) are shown schematically below the autoradiogram.
Molecular Cell 2000 6, DOI: ( /S (00) )