Journal: Nucleic Acids Research

Article Title: Human histone pre-mRNA assembles histone or canonical mRNA-processing complexes by overlapping 3′-end sequence elements

doi: 10.1093/nar/gkac878

Figure Lengend Snippet: The 3′-UTR of histone H2AC18 pre-mRNA can assemble both histone and canonical 3′-mRNA-processing complexes. ( A ) Western blot analysis of complexes purified from HEK293-F nuclear extract (N.E.) with the H2A_4m RNA, prepared with different oligonucleotide concentrations, in the absence or presence of different control molecules (U7, aU7 + SL) or in the presence of the recombinant N-terminal FLASH (N-FLASH) (left subpanel); comparison of results obtained with HEK293-F and HeLa-S3, both using 40 nM H2A_4m (right subpanel). ( B ) Activity assay with a double-biotin wild-type (wt) H2AC18 3′-UTR sequence (structure on top), performed either in crude HEK293-F nuclear extract or with purified histone RNA-processing complex; cleavage products were purified and analyzed by northern blot and luminescent biotinylation detection; complex assembly and cleavage were blocked in the presence of aU7/SL oligonucleotides; the uncleavable GA-H2A was used as weight standard. ( C , D ) Differential analyses of protein abundances measured by MS-based quantitative proteomics: [H2A_4m] versus [H2A_4m + U7/SL] samples (C); [H2A_4m] versus [H2A_4m + aU7/SL] (D). The volcano plots represent the –log 10 (limma P -value) on the y -axis plotted against the log2(fold change) on the x -axis. Relevant identified proteins that are at least 2-fold more abundant with [H2A_4m] compared with [H2A_4m + U7/SL] or [H2A_4m + aU7/SL] are annotated in the zoom-in graphs (subpanels on the left); schematic representations of complex relative abundances are shown (lower subpanels). Abbreviations: CFIm, ceavage factor 1m module (including NUDT21, CPSF6 ands CPSF7 subunits); CPSF, cleavage and polyadenylation specificity factor; CstF, cleavage-stimulating factor module (including CSTF1, CSTF2, CSTF2T and CSTF3 subunits); FLASH, FLICE-associated huge protein; HCC/hCF, histone cleavage complex (SYMPK, CPSF100 and CPSF73 subunits)/human cleavage factor; hPSF, human polyadenylation specificity factor (including CPSF30, CPSF160, WDR33 and FIP1 subunits); PPase, phosphatase module (including SSU72, WDR82 and PP1CA/B/C/R10 subunits); SLBP, stem–loop-binding protein; SYMPK, Symplekin; U7 snRNP, U7 small nuclear ribonucleoprotein (including, besides U7 snRNA, LSM11, LSM10, SNRPD3/B/E/F subunits and the SNRRPG subunit not detected in our analysis).

Article Snippet: CPSF30 (A301-585A), CPSF73 (A301-091A), CPSF160 (A301-580A), SYMPK (A301-465A), SLBP (A303-968A), CstF-64 (A301-092A) and SmD3 (A303-954A) rabbit primary antibodies were purchased from Bethyl Laboratories.

Techniques: Western Blot, Purification, Control, Recombinant, Comparison, Activity Assay, Sequencing, Northern Blot, Quantitative Proteomics, Binding Assay