1. Volume 24, Issue 6, Pages 903-915 (December 2006)
Differential Recruitment of the Splicing Machinery during Transcription Predicts Genome-Wide Patterns of mRNA Splicing 
Michael J. Moore, Elissa M. Schwartzfarb, Pamela A. Silver, Michael C. Yu 
Molecular Cell 
Volume 24, Issue 6, Pages (December 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
The Spliceosome Is Recruited Specifically to Intron-Containing Genes
(A) The distributions of ICGs within genome binding profiles are depicted for the indicated factors. Each green vertical mark represents one ICG. Profiles are plotted against the horizontal axis to indicate the total number of bound genes, as defined in Experimental Procedures. Genes are ranked from left to right by the ratio IP/WCE, which is indicative of binding strength. Intron bias is represented by the degree to which ICGs cluster to the “most bound” end of the profile. ∗From Yu et al. (2004).
(B) Number ICGs bound by each factor is plotted.
(C) Hierarchical clustering of ICG binding profiles is shown.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Early Steps in Spliceosome Assembly Are Transcription Dependent
(A) Double-log plots comparing binding ratio (IP/WCE) to transcriptional frequencies for ICGs are shown with linear regressions and corresponding p values.
(B) Slopes and p values from linear regressions as in (A) are plotted for each factor to represent transcription dependence.
(C) Whole-genome binding profiles as in Figure 1A are shown for Prp40 in 6AU-treated and -untreated cells (upper panel). The lower panel plots number ICGs bound.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Genomic Associations of the Spliceosome Are RNA Dependent
(A) Prp40 and Mud2 genome localizations were analyzed with and without RNase treatment. Numbers of bound ICGs and intronless genes are plotted on the vertical axis.
(B) Prp40 genome localization was determined in spt5-194 and Δcbc cells. Number ICGs bound are plotted on the vertical axis. Differences in the Prp40 WT controls in these two experiments arise from the fact that Prp40 was tagged differently, as noted.
(C) Overlap of Spt5 and Prp40 binding profiles are shown as a Venn diagram. Numbers of genes in corresponding sets are shown. P values derive from Fisher's exact test.
(D) An interaction between Prp40-myc and Spt5-HA was verified by coimmunoprecipitation. α-myc IPs in lanes 2 and 3 show a single band corresponding to Spt5-HA detected by α-HA western blotting (WB). Whole-cell extracts (WCE) show at least two bands corresponding to Spt5, likely representing different phosphorylation states (lanes 4 and 5). No Spt5 is bound in a mock (M) IP. Western analysis with α-PGK1 controlled for IP specificity and gel loading. PGK1 (MW= 44 kDa) is in WCE, but not in α-myc IPs. Bands in lanes 2 and 3 of the α-PGK1 Western are mouse heavy-chain IgG. MW standards are shown to the right of the blot.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Sub2 Controls Early and Late Steps in Spliceosome Assembly
(A) Genomic localization of U2 snRNP protein Lea1 and U5 snRNP protein Brr2 were determined in SUB2-WT and sub2-85 cells. Total bound ICGs are plotted on the vertical axis. Numbers above bars indicate percentage of ICGs as a proportion of total genes bound.
(B) Pairwise comparisons of the Sub2 genomic binding profile to other factors. ∗From Yu et al. (2004).
(C) ChIPs against Mud2 in SUB2-WT and sub2-85 cells were analyzed by quantitative PCR using indicated primer sets in ACT1, RPL30, and PMA1. Genes are schematized with exons in black, introns in white, and numbers indicating nucleotide position relative to translation start site. Values for each primer set are reported as fold enrichment over signal from a primer set amplifying an intergenic region. Error bars indicate the standard deviation of three independent measurements.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Differential Spliceosome Recruitment Predicts Regulated Splicing
(A) Meiotically regulated transcripts were measured by quantitative PCR as described in Experimental Procedures. Product sizes were verified by agarose gel electrophoresis with MW standards and are listed in Table S2 along with primer sequences. Unspliced (U), predicted spliced (S), and nonannotated spliced variants (V) are indicated to the right. ECM33, an ICG strongly bound by the spliceosome, was a control. Triplicate reactions from one experiment are shown.
(B) Analysis as in (A) is shown for non-meiosis-related ICGs.
(C) Quantification of unspliced mRNA accumulation in (B) is shown. Products were normalized to ADH3. Plotted values are average ratios of unspliced to spliced products from triplicate reactions of biological duplicates. Error bars show standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
mRNA Surveillance Machinery Is Recruited to Sites of Regulated Splicing
(A) Binding profiles as in Figure 1A are shown for nuclear exosome components Rrp6 and Lrp1 and nuclear pre-mRNA retention factor Mlp1 (Hieronymus et al., 2004; Casolari et al., 2005). Developmentally regulated ICGs are shown to the right.
(B) Pairwise comparisons of Rrp6, Mlp1, Npl3, and Yra1 are depicted as a Venn diagram. Numbers of genes in each set are shown with pairwise p values determined by Fisher's exact test. This network correlated negatively to hnRNPs Nab2 and Hrp1.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 An Integrated Model of Splicing in mRNP Biogenesis
(A) U1 snRNP and Mud2 sample nascent mRNAs and are retained specifically at ICGs. Roughly 20% of introns are not recognized cotranscriptionally. Of this subset, most are spliced posttranscriptionally, leading to elevated unspliced intermediates that are degraded by the exosome. The exception is meiotically regulated transcripts, which are not efficiently spliced in nonsporulating cells.
(B) Sub2 is recruited to ICGs by Mud2 and then displaces Mud2 to facilitate U2 snRNP recruitment and downstream events. Contacts between RNA Pol II and U1 snRNP mediated by Prp40 may stabilize spliceosomal interactions with the transcription machinery.
(C) Npl3 association with nascent mRNAs facilitates surveillance by the nuclear exosome, which degrades aberrantly spliced mRNAs and promotes patterns of regulated splicing. Rrp6 may function cooperatively with Mlp1 to prevent export of unspliced mRNAs.
(D) Most splicing events complete posttranscriptionally.
(E) Differential mRNA processing may specify recruitment of distinct export factors. We propose Sub2-defined requirements for the export of spliced transcripts and a distinct pathway for transcripts monitored by the exosome.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions