Journal: Genes & Development

Article Title: Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33

doi: 10.1101/gad.250985.114

Figure Lengend Snippet: Reconstitution of AAUAAA-dependent polyadenylation by coexpression of six polypeptides. Three different combinations of CPSF subunits, all including Flag-CPSF160, were expressed in a baculovirus system and purified by Flag affinity purification. ( A ) The main eluate fractions of the three preparations were examined by Western blot to verify the presence of the expected proteins. All signals are from the same blot that was stripped several times and probed separately with the antibodies indicated. All signals for the same subunit are from the same exposure. hFip1 migrated more slowly in the middle lane because the protein carried a Strep tag in this preparation but not in the six-subunit preparation. Silver-stained gels of the same fractions are displayed in Supplemental Figure 1. Viruses used for expression are listed in Supplemental Table 4A. (4c) Four-subunit complex (CPSF160, CPSF100, CPSF73, and CPSF30); (5c) 4c plus hFip1; (6c) 5c plus WDR33. ( B ) Polyadenylation assays were carried out with the same CPSF (sub)complexes shown in A and wild-type L3pre RNA ( left ) or a mutant control ( right ). Increasing amounts of the purified complexes indicated (0.5, 1, 2, and 4 µL of the respective fractions) were incubated with poly(A) polymerase and substrate RNAs as described in the Materials and Methods. Controls with either RNA included an “RNA-only” reaction, two with 1× or 10× poly(A) polymerase in the absence of CPSF, and one with 1× poly(A) polymerase plus CPSF purified from calf thymus (CPSF IV), as indicated. The very weak polyadenylation activity seen with the five-subunit assembly was not AAUAAA-specific; this is difficult to see in this experiment because RNA recovery was lower in the corresponding mutant sample. The difference in poly(A) tail length between the reactions containing calf thymus versus recombinant CPSF should be disregarded, as the tail length obtained in this type of assay depends on CPSF concentration and other variables. ( C ) The same three protein preparations as in A and B were used in nitrocellulose filter-binding assays with 1.5 nM labeled W10 (wild type [wt] or mutant) RNA as described in the Materials and Methods.

Article Snippet: Rabbit antibodies against human WDR33 (no. A301-152A) and symplekin (no. A301-465A) were from Bethyl Laboratories.

Techniques: Purification, Affinity Purification, Western Blot, Strep-tag, Staining, Expressing, Mutagenesis, Control, Incubation, Activity Assay, Recombinant, Concentration Assay, Binding Assay, Labeling

Journal: Genes & Development

Article Title: Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33

doi: 10.1101/gad.250985.114

Figure Lengend Snippet: Identification of mPSF, a CPSF subcomplex active in polyadenylation. Three pairs of CPSF subunits (Flag-CPSF160 and CPSF30; CPSF100 and Strep-CPSF73; and MycHis 6 -WDR33 and hFip1) were expressed and affinity-purified. ( A ) The polypeptide composition of the three preparations was analyzed in silver-stained SDS–polyacrylamide gels. The main eluate fractions are shown. Molecular weights of markers are indicated. Protein identities were verified by Western blotting. All Western blot signals were from the same membrane. Viruses used for expression are listed in Supplemental Table 4B. ( B ) The preparations shown in A were used for polyadenylation assays either separately or in combinations. For the pairwise combinations of subcomplexes, different ratios (1 μL:2 μL, 2:2, and 2:1) were used. RNA substrates were L3pre wild type (wt) or mutant as indicated. Controls (first four lanes for each RNA) were as in .

Article Snippet: Rabbit antibodies against human WDR33 (no. A301-152A) and symplekin (no. A301-465A) were from Bethyl Laboratories.

Techniques: Affinity Purification, Staining, Western Blot, Membrane, Expressing, Mutagenesis

Journal: Genes & Development

Article Title: Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33

doi: 10.1101/gad.250985.114

Figure Lengend Snippet: Characterization of mPSF activities. CPSF160, CPSF30, WDR33, and hFip1 were expressed from a single virus (Supplemental Table 4C) and purified to near homogeneity (see the Materials and Methods). ( A ) The eluate of the final Flag affinity column was analyzed on two Coomassie-stained SDS–polyacrylamide gels. Molecular weights of markers (M) are indicated. The identities of protein bands were verified by Western blotting. All Western blot signals shown were obtained from a single gel lane. ( B ) The preparation shown in A was used for polyadenylation assays. For every 20-µL reaction, 80 fmol of L3preA 15 wild-type (wt) RNA, 80 fmol of mPSF, 1200 fmol of PABPN1 and 8 or 80 fmol of poly(A) polymerase were used as indicated. After preincubation, reactions were started by ATP addition and stopped after the times indicated. In the first lane between the two marker lanes, only RNA was loaded. ( C ) Filter-binding assays were carried out with the Flag eluate and 1.5 nM W10 wild-type or mutant RNAs as described in the Materials and Methods. ( D ) Gel shift assays were carried out with the Flag eluate and 5 nM L3pre or L3preΔ RNA as described in the Materials and Methods. Purified calf thymus CPSF (CPSF IV) was used as a positive control.

Article Snippet: Rabbit antibodies against human WDR33 (no. A301-152A) and symplekin (no. A301-465A) were from Bethyl Laboratories.

Techniques: Virus, Purification, Affinity Column, Staining, Western Blot, Marker, Binding Assay, Mutagenesis, Gel Shift, Positive Control

Journal: Genes & Development

Article Title: Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33

doi: 10.1101/gad.250985.114

Figure Lengend Snippet: WDR33 and CPSF30 can be cross-linked to AAUAAA-containing RNA. ( A ) Ten nanomolar mPSF containing MycHis 6 -WDR33 was incubated with 10 nM radiolabeled 5-iodo-modified iW10 RNA (wild type [wt] or mutant) or unmodified W10 RNA (wild type or mutant), UV cross-linked at 312 nm, and wild-type samples were used for a pull-down with Ni-NTA beads under denaturing conditions. Lanes labeled “load” contain 5% of the samples used for the pull-down. Lanes labeled “E” show 50% of the bead eluates. The first two lanes are no-protein controls (−). Lanes were cut from a single gel and rearranged. ( B ) Ten nanomolar mPSF was incubated with 10 nM radiolabeled iW10 RNA (wild type only), UV cross-linked at 312 nm, and used for a pull-down with antibody directed against CPSF30 or preimmune serum (see the Materials and Methods). “Load” contains 5% of the cross-link reaction, and “E” contains 50% of the eluate. ( C ) Ten nanomolar mPSF containing MycHis 6 -WDR33 was incubated with 10 nM radiolabeled iW10 RNA (wild type only), UV cross-linked at 312 nm, brought to 0.75 M urea, and digested with protease Lys-C as described in the Materials and Methods. Aliquots were taken at the time points indicated, and protein fragments were purified via Ni-NTA beads as in A . “Load” contains 10% of the cross-link reaction, and “E” contains 100% of the eluate.

Article Snippet: Rabbit antibodies against human WDR33 (no. A301-152A) and symplekin (no. A301-465A) were from Bethyl Laboratories.

Techniques: Incubation, Modification, Mutagenesis, Labeling, Purification

Journal: Genes & Development

Article Title: Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33

doi: 10.1101/gad.250985.114

Figure Lengend Snippet: Transcriptome-wide binding of WDR33 to AAUAAA. ( A ) Average density of PAR-CLIP reads around the 1000 most abundantly used CP sites in HEK293 cells. WDR33 shows strong and specific positioning upstream of the CP sites, with a peak at nucleotides −16 to −18 upstream of the cleavage site. ( B ) Enrichment of T-to-C transition relative to the T nucleotide frequency as a function of distance with respect to the poly(A) signal. The analysis is based on the 500 most frequently used CP sites that have a single AATAAA ( top panel) or ATTAAA ( bottom panel) motif and no other variant poly(A) signal in the 40-nt region upstream of the cleavage site. These results indicate that WDR33 is most frequently cross-linked on U nucleotides that immediately follow the polyadenylation signal.

Article Snippet: Rabbit antibodies against human WDR33 (no. A301-152A) and symplekin (no. A301-465A) were from Bethyl Laboratories.

Techniques: Binding Assay, Variant Assay

Journal: Genes & Development

Article Title: Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33

doi: 10.1101/gad.250985.114

Figure Lengend Snippet: Model depicting the polyadenylation complex. WDR33 and CPSF30 are shown binding to the AAUAAA signal. Poly(A) polymerase (PAP) is bound to the 3′ end. hFip1 is shown between AAUAAA and the polyadenylation site; experimentally, binding sites have been mapped both upstream of and downstream from AAUAAA ( ; ; ). CPSF160 is shown binding RNA in an upstream position for reasons discussed in the text. With the exception of WDR33 and CPSF30, which bind the same sequence element but are not known to interact, direct interactions have been reported for all other subunits shown touching each other. Interaction of hFip1 with CPSF30 and CPSF160 has been observed by . Evidence for all other interactions is discussed in the text. The CPSF100–CPSF73–symplekin complex is shown separately; while the complex is clearly part of CPSF, to the best of our knowledge, it is unknown how it associates with mPSF. During the transition from cleavage to polyadenylation, CPSF73, as the endonuclease, has to trade places with poly(A) polymerase.

Article Snippet: Rabbit antibodies against human WDR33 (no. A301-152A) and symplekin (no. A301-465A) were from Bethyl Laboratories.

Techniques: Binding Assay, Sequencing