1. Involvement of SR Proteins in mRNA Surveillance
Zuo Zhang, Adrian R. Krainer 
Molecular Cell 
Volume 16, Issue 4, Pages (November 2004)
DOI: /j.molcel

2. Figure 1
In-Frame Stop Codons Affect Selection of a Duplicated 5′ss In Vivo, but Not In Vitro
(A) Experimental design to test whether in-frame stop codons affect 5′ss selection. 5′ssD is a human β-globin derivative with an internal duplication spanning the 5′ss in the first intron and short flanking exonic and intronic segments. Different in-frame stop codons were placed between the two 5′ss (and after the proximal 5′ss, to keep both 5′ss in exactly the same context) to generate three different constructs: mut1, 2, and 3. The position of each mutation on the pre-mRNA is indicated by an asterisk. For each mutant construct, mRNA spliced via the proximal 5′ss has a PTC.
(B) In vitro splicing of the wild-type (wt) and mutant 5′ssD transcripts with HeLa nuclear extract alone (lanes 1–5) or supplemented with 12 pmol of recombinant SF2/ASF (lanes 6–10), 4 pmol of SF2/ASF lacking the RS domain (lanes 11–15), or 12 pmol of SC35 (lanes 16–20). Wt β-globin without a 5′ss duplication was included as a control (lanes 1, 6, 11, and 16). The positions of the pre-mRNA, and mRNAs spliced via the proximal or distal sites are indicated.
(C) In vivo splicing of wt and mutant 5′ssD, with or without overexpression of SF2/ASF. HeLa cell total RNA recovered after transient expression was analyzed by RPA, with a probe spanning the first exon, the duplicated region, and partial intron 1 sequences. Equivalent results were obtained with probes derived from the wt or any of the mutant constructs. A GFP plasmid was cotransfected as an internal reference for transfection efficiency, RNA recovery, and loading. β-globin and GFP probes were used simultaneously; undigested probes (P) are shown in lane 1, corresponding to <1% of the amount of probe used in each RPA reaction.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Differential Use of 5′ss In Vivo Requires Overexpression of SF2/ASF with a Functional RS Domain
Transient transfections were done with wt and mutant 5′ssD, with or without overexpression of the indicated versions of SF2/ASF. SF2-ΔRS, a truncated form lacking the RS domain; SF2-RS10, a variant in which the natural RS domain was replaced by ten RS dipeptides. The transcripts were analyzed by RPA, as in Figure 1C. A GFP plasmid was cotransfected and its transcripts assayed as an internal control. M, DNA size markers.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Effect of Different SR Proteins on Alternative 5′ss Selection and Sensitivity to a PTC
(A) Transient transfections with wt and mut2 5′ssD, with overexpression of the indicated SR proteins. The globin and control GFP transcripts were analyzed by RPA as in Figure 1C.
(B) Western blot to measure the various overexpressed SR proteins via a common T7-epitope tag. Endogenous β-catenin served as an internal control.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
The Differential Use of Alternative 5′ss between Wt and Mutant Transcripts Is Due to NMD, Rather than NAS
(A) Transient transfections of 5′ssD constructs under conditions of siRNA-mediated knock-down of hUpf1 or hUpf2. Transfections were carried out 48 hr after siRNA treatment (Mendell et al., 2002). Total cell RNA was harvested after another 36 hr and analyzed by RPA. The relative amount of mutant to wt proximal spliced mRNA under each condition, normalized to the control GFP mRNA level, is shown at the bottom.
(B) Western blot showing the attenuation of endogenous hUpf1 and hUpf2 protein levels by siRNA treatment. Endogenous hnRNP A1 was measured as a control.
(C) Transient transfections with cycloheximide treatment (lanes 5 and 6) or coexpression of wt or dominant-negative (R844C) forms of hUpf1 (lanes 7–10).
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Overexpression of SR Proteins Enhances β-globin NMD
(A) Levels of transiently expressed β-globin transcripts with or without a PTC at codon 39 in exon 2, as a function of increased coexpression of various SR proteins or hnRNP A1. Transiently expressed GFP and β-globin transcripts were detected by RPA as in Figure 1C. The globin probe spans exon 1 and part of intron 1. The quantitation of wt and PTC-containing (Ter39) mRNA under each condition—normalized to the control GFP mRNA level—is shown below each lane. We obtained equivalent results with a different probe spanning exon 2 and intron 2 (not shown).
(B) Western blot showing the levels of the overexpressed proteins or endogenous β-catenin.
(C) RPA shows that the −16 cryptic spliced β-globin mRNA can be targeted to the NMD pathway by overexpression of SF2/ASF. HeLa cells were transiently transfected with wt and various mutant β-globin constructs, with (lanes 9–20) or without (lanes 3–8) overexpression of SF2/ASF, and total cell RNA was harvested after 36 hr. Dominant-negative Upf1 (R844C) was overexpressed to inhibit NMD (lanes 15–20); wt Upf1 served as the control (lanes 3–14). The positions of spliced mRNAs corresponding to the wt or −16 cryptic 5′ss are indicated.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 Overexpression of SR Proteins Enhances GPX1 NMD
(A) Levels of GPX1 transcript after transient transfection into COS cells, measured by RPA with a probe spanning part of the last exon and the last intron. The ratio of mutant to wt mRNA under each condition, normalized to the control GFP mRNA level, is shown below each lane.
(B) Western blot of the overexpressed SR proteins or endogenous β-catenin.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
The Effect of SF2/ASF on NMD Does Not Correlate with Shuttling
(A) Transient transfections with wt and mutant 5′ssD, with overexpression of SF2/ASF or derivatives of it that are retained in the nucleus. RS35, RS domain from SC35; NRS, nuclear retention signal from SC35. Transcripts were assayed by RPA as in Figure 1C. The quantitation of proximal spliced mRNA—normalized to the control GFP mRNA level—is shown below each lane.
(B) The same SF2/ASF derivatives were tested in transient transfections with a standard β-globin NMD substrate with a PTC in codon 39. Transcripts were assayed by RPA as in Figure 5A. The quantitation of wt and PTC-containing (Ter39) mRNA—normalized to the control GFP mRNA level—is shown below each lane.
Molecular Cell  , DOI: ( /j.molcel )