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. 2013 Apr 25;3(4):1080–1092. doi: 10.1016/j.celrep.2013.03.012

Figure 1.

Figure 1

Isolation of Nucleoplasmic CstF-64-Associated Pre-mRNA

(A) Diagram of the CoTC termination mechanism. The CoTC terminator (red bar) is located downstream of the poly(A) site (pA, green arrowhead) of the human β-globin gene. β-globin pre-mRNA (curved black line with blue cap) is generated by Pol II (gray icons). 3′ flanking region transcripts (red dashed line) are cleaved by CoTC activity (scissors). Xrn2 (blue icon) degrades the 3′ transcript (black dashed line) leading to Pol II and pre-mRNA release from the DNA template. Pre-mRNA is processed by poly(A) factors including CstF-64 (gray sphere) in the nucleoplasm to mRNA (line with AAAA indicating poly(A) tail).

(B) RT-PCR analysis of immunoprecipitated nucleoplasmic RNA. RNA in control input (lanes 1 and 4) and immunoprecipitated with rabbit IgG (lanes 2 and 5) or CstF-64 antibody (lanes 3 and 6) was subjected to RT-PCR analysis using β-globin-specific primers (blue arrows in diagram beside the data panel) and primers for 7SK snRNA. The lack of bands in lanes 4–6 (−RTase) confirms the absence of contaminating DNA in all samples.

(C) Purification of CstF-64-RNA covalent complexes by SDS-PAGE following treatment with high (40 U/ml, lanes 1–4) or low (4 U/ml, lanes 5–8) RNaseI. No protein-RNA complexes were detected in the absence of UV irradiation (lanes 1, 2, 5, and 6) or following control immunoprecipitation with rabbit IgG (lanes 3 and 7), demonstrating the specificity of the CstF-64 antibody/CstF-64-RNA complex interaction. The red dashed box, in lane 8, indicates the area of the gel from which CstF-64-RNA complexes were eluted for subsequent HITS analysis.

(D) Bar graphs showing the distribution of CLIP reads in annotated genic and intergenic regions (left panel) and in intron, exon, and 3′ UTRs of annotated genes (right panel). P values (brackets) for the significance of relative enrichment, with respect to what would be expected by chance, in the case of a random distribution, were calculated using a one-sided binomial test. ∗Indicates significant enrichment (p value < 0.001).

(E) Diagram of the human β-globin gene with CLIP tags (dark blue squares) mapped to the sequence shown below the diagram. The annotated poly(A) site is indicated in green. Red letters highlight G/U stretches representing potential CstF-64 binding sites.