1. Volume 7, Issue 1, Pages 227-232 (January 2001)
Specific Alterations of U1-C Protein or U1 Small Nuclear RNA Can Eliminate the Requirement of Prp28p, an Essential DEAD Box Splicing Factor 
Jeff Yi-Fu Chen, Leah Stands, Jonathan P Staley, Ronald R Jackups, Lori J Latus, Tien-Hsien Chang 
Molecular Cell 
Volume 7, Issue 1, Pages (January 2001)
DOI: /S (01)

2. Figure 1 PRP28 Can Be Bypassed by Extragenic Mutations
(A) Growth phenotypes of suppressor strains. Relevant genotypes of yeast strains are shown on the left, and their growth phenotypes (+, growth; −, no growth) in tryptophan (Trp)-minus medium are indicated to the right. Cells were grown to saturation at 30°C, serially diluted, and spotted onto YPD (1% yeast extract, 2% peptone, and 2% dextrose) plates. A set of three plates was separately incubated at 30°C, 25°C, and 16°C.
(B) Restriction maps of the chromosomal PRP28, prp28Δ::HIS3 (HIS3), and the plasmid-borne prp alleles. The plasmid (pCA8070) is marked by the TRP1 selectable marker. The probe used in (C) is a 2.8 kb NheI–EcoRV DNA fragment.
(C) Genomic analysis of the suppressor strains. Genomic DNAs isolated from the wild-type (PRP28) strain, Trp+ (+; lanes 2, 4, and 6) and Trp− (−; lanes 3, 5, and 7) suppressor strains (SUP1, SUP2, and SUP13) were digested by EcoRV and analyzed by DNA blotting using the probe shown in (B).
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2 Splicing Proceeds Normally in the Bypass Suppressor Strains
(A) Splicing in vivo. Splicing of the actin transcript was monitored in PRP28 (lanes 1 and 4), prp (lanes 2 and 5), and prp28Δ::HIS3 SUP1 (lanes 3 and 6) strains using a 32P-labeled oligonucleotide hybridizing to exon 2 of the actin pre-mRNA for primer extension. The cDNA products representing the actin mRNA and pre-mRNA are indicated on the right. The lengths (nt, nucleotide) of the DNA markers are indicated on the left. Total RNA was prepared from cultures that had been grown at 30°C to 0.5 OD600 units before shifting them to 16°C for 16 hr.
(B) Splicing in vitro. Standard splicing reactions (Lin et al. 1985) using a 32P-labeled actin transcript as a substrate were incubated at 25°C for 20 min (lanes 7 and 9) or 16°C for 1 hr (lanes 8 and 10). Splicing extracts were made from PRP28 (lanes 7 and 8) and prp28Δ::HIS3 SUP1 (prp28Δ SUP1; lanes 9 and 10) strains. The positions of lariat intermediate, lariat intron, pre-mRNA, mRNA, and exon 1 are indicated by symbols on the right.
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Sequence Alignment of the Evolutionarily Conserved U1-C Proteins
U1-C proteins are most conserved in their N termini (top thick line), which include a conserved C2H2-type zinc finger (*). The L13F (SUP1 and SUP2) and L13S (SUP13) mutations found in bypass suppressor strains are shown. (Hs, H. sapiens; Mm, M. musculus; Xl, X. laevis; Ce, C. elegans; Sp, S. pombe; Sc, S. cerevisiae.)
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4 Weakening the U1/5′ SS Interaction Can Bypass Prp28p
(A) Base pairing between U1 snRNA and the pre-mRNA 5′ SS. U1 snRNA is numbered from its 5′ end. Exons in the pre-mRNA are boxed.
(B) Mutations in U1 snRNA can bypass Prp28p. Growth at 30°C on the 5-FOA plate of strains containing various U1 mutant clones is shown. Note that serial dilutions of the cell cultures are shown from the top down for easier correlation with the numbered U1 snRNA depicted in (A).
(C) The L13S substitution in U1-C suppresses hyperstabilization of the U1 snRNA/5′ SS interaction. Growth at 25°C in the presence of 0.5 mM copper (Cu2+) is shown for yeast containing wild-type or L13S-substituted U1-C, the chromosomal copy of U1-C, and an ACT1-CUP1 fusion reporter having a wild-type 5′ SS or mutated 5′ SS that forms ten contiguous base pairs with U1 snRNA. Growth at higher copper concentrations reflects greater splicing efficiency.
Molecular Cell 2001 7, DOI: ( /S (01) )