%
Check for updates
Molecular basis for the recognition of the human AAUAAA polyadenylation signal
Yadong Sun3'1, Yixiao Zhangb'1, Keith Hamilton3, James L. Manley3,2, Yongsheng Shic, Thomas Walzb'2, and Liang Tong3,2
aDepartment of Biological Sciences, Columbia University, New York, NY 10027; bLaboratory of Molecular Electron Microscopy, Rockefeller University, New York, NY 10065; and department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697
Contributed by James L. Manley, November 14, 2017 (sent for review October 26, 2017; reviewed by Nick J. Proudfoot and Joan A. Steitz)
Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3'-end for maturation. A crucial step in this process is the recognition of the AAUAAA polyadenylation signal (PAS), and the molecular mechanism of this recognition has been a long-standing problem. Here, we report the cryo-electron microscopy structure of a quaternary complex of human CPSF-160, WDR33, CPSF-30, and an AAUAAA RNA at 3.4-A resolution. Strikingly, the AAUAAA PAS assumes an unusual conformation that allows this short motif to be bound directly by both CPSF-30 and WDR33. The A1 and A2 bases are recognized specifically by zinc finger 2 (ZF2) of CPSF-30 and the A4 and A5 bases by ZF3. Interestingly, the U3 and A6 bases form an intramolecular Hoogsteen base pair and directly contact WDR33. CPSF-160 functions as an essential scaffold and preorganizes CPSF-30 and WDR33 for high-affinity binding to AAUAAA. Our findings provide an elegant molecular explanation for how PAS sequences are recognized for mRNA 3'-end formation.
pre-mRNA 3'-end processing | polyadenylation | RNA recognition | zinc finger | WD40 domains
In eukaryotes, messenger RNAs are transcribed from the genome by RNA polymerase II (Pol II). However, these primary transcripts (pre-mRNAs) must undergo extensive processing, including 5'-end capping and splicing. The pre-mRNAs must also be processed at their 3' end. For most of them, the processing involves an endonucleolytic cleavage at a specific location followed by the addition of a poly(A) tail (polyadenylation) (1-5). Metazoan replication-dependent histone pre-mRNAs are distinct in that they are only cleaved but not polyadenylated (6, 7). The mature mRNAs are then transported into the cytoplasm, where they can be translated into proteins.
A large number of protein factors have been identified for pre-mRNA 3'-end processing (8-10). The machinery for canonical 3'-end processing (cleavage and polyadenylation) has been fractionated into several components based on classical biochemistry experiments, including the cleavage and polyadenylation specificity factor (CPSF) (11, 12) and the cleavage stimulation factor (CstF) (13, 14) in mammals. CPSF has six subunits and has central roles in 3'-end processing. Its CPSF-73 subunit is the endoribonuclease for the cleavage reaction (15), and its WDR33 and CPSF-30 subunits are required for recognizing the polyadenylation signal (PAS) in the pre-mRNA (16, 17), which helps define the position of RNA cleavage. The PAS consists of a hexanucleotide, most frequently with the sequence AAUAAA (18, 19) and first identified more than 40 y ago (20), and is typically located 10-30 nucleotides upstream of the cleavage site. CstF has three subunits and recognizes a G/U-rich sequence motif downstream of the cleavage site. It also contributes to the selection of the cleavage site, including alternative polyadenylation (21-23).
The subunits of CPSF form two functional components. mPSF (mammalian polyadenylation specificity factor) consists of CPSF-160, WDR33, CPSF-30, and Fipl, and is necessary and sufficient for PAS recognition and polyadenylation [with poly(A)
www. pnas.org/cgi/doi/10.1073/pnas. 1718723115
polymerase] (17). CPSF-73 and CPSF-100 together with sym-plekin form the other component, also known as the core cleavage complex (24) or mCF (mammalian cleavage factor) (5), which catalyzes the cleavage reaction. Symplekin is a scaffold protein and also mediates interactions with CstF and other factors in the 3'-end processing machinery (25-28).
CPSF-160 (160 kDa) contains three p-propeller domains (BPA, BPB, and BPC) and a C-terminal domain (CTD) (Fig. L4), and shares weak sequence homology to the DNA damage-binding protein (DDB1) (29, 30). WDR33 (146 kDa) contains a WD40 domain near the N terminus and a collagen-like segment near the middle (10). CPSF-30 (30 kDa) contains five CCCH zinc-finger motifs (ZF1-ZF5) near the N terminus and a CCHC zinc knuckle at the C terminus (31).
The recognition of the PAS is a critical step in canonical 3'-end processing. However, its molecular mechanism is not known, and this has been a long-standing question in the field. Here, we have determined the structure of a quaternary complex of human WDR33, CPSF-30, CPSF-160, and a 17-mer AAUAAA PAS RNA by cryogenic electron microscopy (cryo-EM) at 3.4-A resolution. Most importantly, the structure has iUuminated the molecular basis for the specific recognition of the PAS, revealing direct roles of CPSF-30 and WDR33, as well as extensive interactions
Author contributions: Y. Sun, Y.Z., K.H., Y. Shi, T.W., and L.T. designed research; Y. Sun, Y.Z., K.H., T.W., and L.T. performed research; Y. Shi contributed new reagents/analytic tools; Y. Sun, Y.Z., J.L.M., T.W., and L.T. analyzed data; and Y. Sun, Y.Z., J.L.M., Y. Shi, T.W., and L.T. wrote the paper.
Reviewers: N.J.P., Sir William Dunn School of Pathology; and J.A.S., Yale University and HHMI.
The authors declare no conflict of interest. Published under the PNAS license.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 6BLL, 6BLY, and 6BM0).
1Y. Sun and Y.Z. contributed equally to this work.
2To whom correspondence may be addressed. Email: jlm2@columbia.edu, twalz@mail. rockefeller.edu, or ltong@columbia.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi: 10. 1073/pnas.1718723115/-/DCSupplemental.
PNAS  |  Published online December 5, 2017  | E1419-E1428
Significance
The AAUAAA polyadenylation signal (PAS) was identified more than 40 years ago, but it has remained a mystery how this signal is recognized at the molecular level, which is required for the 3-end processing of nearly all eukaryotic messenger RNA precursors. We have determined the cryo-electron microscopy structure of a quaternary complex of human CPSF-160, WDR33, CPSF-30, and an AAUAAA RNA at 3.4-A resolution. The AAUAAA PAS assumes an unusual conformation and is recognized directly by both CPSF-30 and WDR33. CPSF-160 functions as an essential scaffold and preorganizes CPSF-30 and WDR33 for high-affinity binding to AAUAAA. Our findings provide an elegant molecular explanation for how PAS sequences are recognized for mRNA 3-end formation.
A CPSF-160
BPA
BPB ~BPC    M1443
WDR33 |      WD40 ~
1336
CPSF-30
269
B CPSF-160-WDR33 + CPSF-30 + RNA
120 -j CPSF-160-WDR33 + CPSF-30 (33-170) CPSF-160-WDR33
103 -I ^^^^^^^^^
CPSF-160 ■^lU^^'J^^ WDR33
13.5 14 14.5    15 15.5 16(mi;
10.0 12.0 Volume (ml)
3.4
3 r>
<
o
CPSF-160
CPSF-160
BPA
80 A
40 A
Fig. 1. Overall structure of the human CPSF-160-WDR33-CPSF-30-PAS RNA quaternary complex. (A) Domain organizations of human CPSF-160, WDR33, and CPSF-30. The p-propeller domains of CPSF-160 are labeled and given different colors, and the CTD in brown. The WD40 domain of WDR33 is in light blue, and the surrounding segments in cyan. The collagen-like segment is in gray. ZF1-ZF5 in CPSF-30 are in green, and the zinc knuckle in gray. (6) Gel filtration profiles of the quaternary complex (red), the CPSF-160-WDR33 binary complex (blue), and a mixture of CPSF-160, WDR33, and a CPSF-30 deletion mutant (light blue). (Inset) SDS/PAGE of the quaternary (migration positions labeled in red) and binary (blue) complex samples. Fractions at the peak of the profiles were used for EM studies. (C) Local resolution map for the CPSF-160-WDR33-CPSF-30-PAS RNA quaternary complex. (D) Local resolution map for the CPSF-160-WDR33 binary complex. (F) Overall structure of the quaternary complex, colored as in A. The PAS RNA is in orange. The molecular surface of the structure is shown as a transparent surface. The top faces of BPA and BPC of CPSF-160 and the WD40 domain of WDR33 are indicated with black arrowheads. (F) Overall structure of the quaternary complex, viewed after a rotation of 90° around the vertical axis. C and D were produced with Chimera (68), and the other structure figures were produced with PyMOL (https://pymol.Org/2/).
E1420  | www.pnas.org/cgi/doi/10.1073/pnas.1718723115
Sun et al.
among the protein factors in this complex, which preorganize the binding site for the PAS RNA.
Results
Structure Determination. We produced the complex of full-length human CPSF-160 with the N-terminal region of human WDR33 (residues 1-572) by coexpressing them in baculovirus-infected insect cells (Fig. IB) and studied the sample by EM. We produced full-length human CPSF-30 with an N-terminal fusion to maltose-binding protein (MBP) by expression in bacteria. To prepare the quaternary complex, the three proteins were mixed together with a 17-mer RNA oligo containing a PAS (5'-AAC-CUCCAAUAAACAAC-3', with the PAS underlined), incubated with TEV protease to remove the MBP, and the complex was purified by gel filtration chromatography for EM (Fig. IB).
We determined the structures of the CPSF-160-WDR33 binary complex at 3.8-A resolution by cryo-EM (SIAppendix, Figs. S1-S3), which allowed us to build an atomic model of the complex. We next determined the structure of the CPSF-160-WDR33-CPSF-30-PAS RNA quaternary complex at 3.4-A resolution (Fig. 1C and SI Appendix, Figs. S2 and S3). Compared with CPSF-160 and WDR33, the resolution of the CPSF-30 and RNA region is somewhat lower, suggesting that these two molecules may have some overall flexibility in the quaternary complex. We also determined a higher quality structure of the CPSF-160-WDR33 binary complex, at 3.36-A resolution, based on the cryo-EM images from the quaternary complex sample (Fig. ID and SI Appendix, Figs. S2 and S3). The atomic models for the three structures have good fit to the EM density as well as expected geometric parameters (SI Appendix, Fig. S3 and Table SI).
Overall Structure of the Quaternary Complex. The overall structure of the quaternary complex has dimensions of 110 A x 80 A x 40 A (Fig. 1E and F). The three p-propellers of CPSF-160 are arranged in a trefoil pattern, and its CTD is located near the center of the trefoil, interacting with the sides of the three propellers. WDR33 is bound between the "top" faces of BPA and BPC The top face is where the loops connecting neighboring blades of a propeller are located (30). CPSF-30 is located on one side of the structure, contacting both BPC in CPSF-160 and WDR33. The PAS RNA is positioned between CPSF-30 and WDR33, and has no direct interactions with CPSF-160.
Twelve surface segments of CPSF-160 (containing 266 of the 1,443 residues) are not included in the atomic model of the quaternary complex due to lack of density, suggesting that they are likely disordered. Residues 1-42 and 418-572 of WDR33 are disordered in the quaternary complex. Residues 117-269 of CPSF-30 are also disordered, which include ZF4, ZF5, and the zinc knuckle. These residues of CPSF-30 may mediate other functions in the pre-mRNA 3'-end processing machinery, although the yeast homolog Ythl does not contain a zinc knuckle.
Interaction Between CPSF-160 and WDR33. CPSF-160 and WDR33 have an extensive interface, burying ~3,700 A2 of the surface area of each protein (Fig. 24). For WDR33, the interaction with CPSF-160 is primarily mediated by the segment before its WD40 domain, residues 52-109, which contribute 2,500 A2 to the buried surface area. These residues are located in a deep pocket at the BPA-BPC interface of CPSF-160 (Figs. IE and IB). The loops on the bottom face of the WD40 domain contribute 850 A2 to the buried surface area, and they contact loops on the top faces of BPA and BPC (Fig. IE). The segment C-terminal to the WD40 domain (residues 403-417) contributes 250 A2, and these residues are located near the segment before the WD40 domain (Fig. 2B). Residues 41-109 and 403-420 are well conserved, while
residues 1-40 are poorly conserved among WDR33 homologs (SI Appendix, Fig. S4).
The interaction between the N-terminal segment of WDR33 and BPC involves residues 52-73 and 85-91 of WDR33 and is more extensive than that with BPA of CPSF-160, which involves residues 74-84 and 99-104 of WDR33. Residues 60-71 of WDR33 form a helix and are positioned near the center of the BPC top face, showing hydrophobic as well as polar interactions (Fig. 2C). The loop containing residues 87-91 has intimate contacts with two blades of BPC, at one side of the top face (Fig. 2C). In contrast, a loop containing residues 72-82 of WDR33 is located near the center of the BPA top face, with two Arg residues (Arg74 and Arg77) projecting into the central cavity of the propeller (Fig. 2D). However, these two residues do not establish strong interactions with BPA, as they do not have clearly favorable binding partners in CPSF-160. The loop containing residues 98-104 of WDR33 is positioned at one side of the top face of BPA, interacting with two blades of the propeller (Fig. 2D).
The large surface area buried at the CPSF-160-WDR33 interface suggests that their binary complex should be stable as well. In fact, the overall structure of the CPSF-160-WDR33 binary complex is essentially the same as that in the quaternary complex (SI Appendix, Fig. S5), with an rms distance of 0.24 A for 1,539 equivalent Cot atoms. Therefore, binding of CPSF-30 and PAS RNA does not cause a global structural change in the CPSF-160-WDR33 complex, except that residues 43-54 of WDR33 are disordered in the binary complex. They become ordered in the quaternary complex and contact the PAS RNA and ZF3 of CPSF-30 (SI Appendix, Fig. S5; see below).
Interaction of CPSF-30 with CPSF-160 and WDR33. The interface between CPSF-160 and CPSF-30 buries 1,700 A2 of the surface area of each protein (Fig. 24). Residues 1-26 of CPSF-30, in an N-terminal extension before the zinc fingers (Fig. L4) and well conserved among homologs (SI Appendix, Fig. S6), contact the bottom face of BPC (Figs. IE and 3A), contributing 1,100 A2 to the buried surface area, indicating that these residues are crucial for the interactions between CPSF-160 and CPSF-30. Residues 1-7 of CPSF-30 are located in a deep pocket at the center of the BPC bottom face (Fig. 3>B). Consistent with the structural observations, we found that a CPSF-30 mutant missing the first 32 residues (as well as C-terminal residues disordered in the quaternary complex structure) could not form a ternary complex with CPSF-160 and WDR33 (Fig. IB).
ZF1-ZF3 of CPSF-30 contact both WDR33 (850 A2 buried surface area) and the BPC of CPSF-160 (600 A2; Fig. 3C). The smaller buried surface area here suggests that the interactions are somewhat weaker, although the PAS RNA is situated between WDR33 and CPSF-30 and is likely to stabilize their interactions. ZF1-ZF2 contacts the side of BPC and the edge of the bottom face of the WDR33 WD40 domain, while ZF3 primarily interacts with the N-terminal segment of WDR33 (residues 43-54) that becomes ordered in the quaternary complex (Fig. 3C).
Binding Mode of PAS RNA. While a 17-mer RNA oligo was used for making the quaternary complex, the cryo-EM map revealed density for only seven nucleotides, the entire AAUAAA PAS and the following nucleotide, which has weak density (Fig. 4^4). The fact that only the AAUAAA of the RNA oligo is well ordered in the structure is consistent with this complex being responsible for recognizing the PAS for pre-mRNA 3'-end processing. The base of the nucleotide following the PAS is exposed to the solvent and has no contact with the proteins, while its ribose is located near the side chain of Asn53 of WDR33.
The AAUAAA PAS is located between the side of the WD40 domain of WDR33 and ZF2-ZF3 of CPSF-30 (Figs. IE
Sun et al.
PNAS  |  Published online December 5, 2017  | E1421
Fig. 2. Interactions between human CPSF-160 and WDR33. (A). Buried surface areas at the interfaces in the CPSF-160-WDR33-CPSF-30-PAS RNA quaternary complex. Curved arrows indicate the major contacts at the CPSF-160-WDR33 and CPSF-160-CPSF-30 interfaces. (B) Molecular surface of CPSF-160, colored by domains. The N- and C-terminal segments of WDR33 beyond the WD40 domain, shown as cartoons in cyan and dark cyan, respectively, are located in a deep pocket in CPSF-160. (C) Interactions between WDR33 (residues 59-73 and 87-91, cyan) and the top face of BPC of CPSF-160 (yellow). Most of the residues shown in sticks contribute >50 A2 to the buried surface area at the interface. (D) Interactions between WDR33 (residues 72-82 and 98-104, cyan) and the top face of BPA of CPSF-160 (salmon).
and AB). The six bases are positioned in clearly defined pockets, while the backbone phosphates are mostly exposed to the solvent, although there are many positively charged residues near the RNA (Fig. AC). Residues 43-54 in the N-terminal region of WDR33 become ordered in the quaternary complex, and cover up part of the RNA, although the density for this segment is relatively weak (Fig. AC).
The backbone of the AAUAAA PAS assumes an S shape, and there are no stacking interactions among the six bases, which are all in the anti conformation (Fig. AB). Interestingly, we found that U3 and A6 form a Hoogsteen U-A base pair (the nucleotides in the PAS are numbered from 1 to 6 here). The six bases in the PAS are arranged such that Al and A2 are pointed in one direction, and are recognized by ZF2 of CPSF-30 (Fig. AB).
E1422  | www.pnas.org/cgi/doi/10.1073/pnas.1718723115
Sun et al.
Fig. 3. Interactions of human CPSF-30 with CPSF-160 and WDR33. (A) Molecular surface of CPSF-160, colored by domains. CPSF-30 is shown as a cartoon in green, and the metal ions in the zinc fingers as spheres (pink). (6) Interactions between CPSF-30 (residues 1-11, green) and the bottom face of BPC of CPSF-160 (yellow). Most of the residues shown in sticks contribute >40 A2 to the buried surface area at the interface. (C) ZF1-ZF3 of CPSF-30 (green) contact the side of BPC of CPSF-160 (yellow) and the bottom face of the WDR33 WD40 domain (light blue). The PAS RNA is in orange.
A4 and A5 are pointed in another direction, perpendicular to that of A1-A2, and these two bases are recognized by ZF3 of CPSF-30. The U3-A6 base pair is pointed in the opposite direction from that of A4-A5, and is bound by WDR33. With this arrangement, the bases of the trinucleotide A2-U3-A4 are splayed apart, and such conformations have been observed in other protein-RNA complexes (32-34).
Specific Recognition of PAS RNA. The structure reveals that Al, A2, A4, and A5 of the PAS are recognized specifically by CPSF-30. The Nl and N6 atoms of the Al base are recognized by hydrogen bonds with the main-chain amide of Lys69 and main-chain carbonyl of Val67 in ZF2, respectively (Fig. AD), a hydrogen-bonding pattern that is identical to that in an A-U Watson-Crick base pair as well as specific recognition of adenine base by other proteins. The Nl atom of the A2 base is recognized by a hydrogen bond with the main-chain amide of Lys77, while its N6 atom does not appear to be recognized. In addition, the Al base is ji-stacked with the side chain of Phe84 on one face and flanked by that of Lys69 on the other (Fig. AD). The A2 base is ji-stacked with the side chain of His70 on one face and surrounded by Lys77 and Lys78 on the other. Both bases are also located near the metal ion and its ligands in ZF2.
Remarkably, the A4 and A5 bases are recognized by ZF3 of CPSF-30 using similar interactions (Fig. AE). In fact, the two zinc fingers show a conserved mechanism of recognizing the bases. An overlay of ZF3 with ZF2 also brings their cognate bases into overlap (Fig. AF). The geometry of these hydrogen bonds is not
Sun et al.
optimal, possibly due in part to the limited resolution of the current structure. It might also be possible that some of these suboptimal interactions are genuine structural features, which would allow some variability in the nucleotides in the PAS. Nonetheless, the hydrogen-bonding pattern clearly indicates that adenines would be preferred at these positions.
The U3-A6 base pair is ji-stacked with the side chain of Phel53 in the WD40 domain of WDR33, while its other face is in contact with residues 43-45 in its N-terminal segment (Fig. AG). Lysll7 and Ilel56 surround the side of this base pair. While these two bases do not appear to be specifically recognized directly, a G6 base would not be preferred here as its 2-amino group would clash with the main-chain carbonyl of Thrll5. Residues that are in contact with the RNA are highly conserved among WDR33 and CPSF-30 homologs (57 Appendix, Figs. S4 and S6).
The recognition mode of the A-A dinucleotide by ZF2 and ZF3 of CPSF-30 is distinct from those in other CCCH zinc fingers, such as that in Nab2 (35), the splicing factor muscleblind (MBNL1) (36, 37), and the AU-rich element binding protein TISlld (38) {SI Appendix, Fig. S7). While the position of the Al base in ZF2 has approximate counterparts in the other structures, the position of A2 in CPSF-30 is unique. In addition, even the direction of the phosphate backbone can be different in the RNAs bound to these ZFs. Overall, there appears to be substantial variability in ribonucleotide recognition by CCCH zinc fingers.
PNAS  |  Published online December 5, 2017  | E1423
Fig. 4. Recognition of the PAS RNA by CPSF-30 and WDR33. (A) Cryo-EM density (magenta mesh) for residues AAUAAAC of the PAS RNA (orange) used in this study, contoured at 4.5o. (B) Binding mode of the AAUAAA PAS (orange) at the interface of CPSF-30 ZF2-ZF3 (green) and WDR33 (light blue forWD40 domain and cyan for N-terminal segment). Hydrogen bonds in the U3-A6 Hoogsteen base pair are indicated with dashed lines in red. (C) Molecular surface of the CPSF-160-WDR33-CPSF-30 complex, colored by electrostatic potential (red, negative; blue, positive). The N-terminal segment of WDR33 (residues 43-52) is shown as a cartoon only. The side chains of Lys46, Arg47, and Lys50 have no density and are shown as lines. The side chain of Arg49 has density and is shown as sticks. PAS RNA is shown as stick model in orange. (D) Detailed interactions showing the recognition of the A1 and A2 bases of the PAS (orange) by ZF2 of CPSF-30 (green). Hydrogen bonds between the adenine bases and the protein are indicated with dashed lines in red. (F) Detailed interactions showing the recognition of the A4 and A5 bases of the PAS (orange) by ZF3 of CPSF-30 (green). (F) Overlay of the structures of ZF2 (green) and ZF3 (gray) of CPSF-30 brings their cognate di-nucleotides (orange and gray, respectively) into overlap as well. (6) Recognition of the U3-A6 base pair (orange) by WDR33 (blue). (H) Overlay of the structure of ZF2-ZF3 of CPSF-30 in the quaternary complex (green) with that in the complex with influenza virus NS1A protein (PDB ID code 2RHK) (43). NS1A is shown in gray, clashing with the PAS RNA.
CPSF-30 and WDR33 Bind PAS RNA Synergistically. Our structure shows that both CPSF-30 and WDR33 are directly involved in PAS recognition, while CPSF-160 is not in contact with this
hexanucleotide. To test the importance of the three proteins in this binding, we carried out electrophoretic mobility shift assays (EMSAs) using the 17-mer RNA oligo, with a 3'-end fluorescent label to aid visualization. We used protein samples that were purified by gel filtration and showed homogeneous, non-aggregated behavior on the column (SI Appendix, Fig. S8) for these assays. Our data showed that the CPSF-160-WDR33-CPSF-30 ternary complex has high affinity for the AAUAAA PAS RNA (Fig. 5/4), while the affinity for the variant AAgAAA RNA is lower (Fig. 5B), consistent with earlier data (17). In contrast, the CPSF-160-WDR33 binary complex showed only weak binding (Fig. 5C), while the MBP-CPSF-30 fusion protein showed essentially no binding to the AAUAAA PAS RNA (Fig. 5D). A mixture of CPSF-160 and MBP-CPSF-30, which did not appear to form a binary complex (SI Appendix, Fig. S8), also had very low affinity for the PAS RNA (Fig. 5E). In contrast, a mixture of the CPSF-160-WDR33 binary complex with a CPSF-30 deletion mutant (containing residues 33-170) that cannot form the ternary complex (Fig. IB) showed appreciable binding for the PAS RNA (Fig. 5F), suggesting that the PAS RNA can stabilize the quaternary complex. Overall, the EMSA data indicate that the affinity of CPSF-30 and WDR33 alone or together with CPSF-160 for the PAS RNA is low, and the two proteins bind synergistically to the PAS RNA in the presence of CPSF-160.
CPSF-160 Is a Scaffold. The overall structure of the CPSF-160-WDR33 complex has similarity to that of the DDB1-DDB2 complex that is important for DNA damage repair (39). The relative positions of BPA and BPC are similar to those in DDB1 (SIAppendix, Fig. S9). Large variations in the positioning of BPB are observed in DDB1, although we did not see significant flexibility in BPB of CPSF-160 in our studies here (Fig. 1C). DDB2 is bound between BPA and BPC of DDB1 as well, also using N-terminal extensions beyond the p-propeller domain. However, the position of the WD40 domain in WDR33 is different from that of DDB2 in the complex. Moreover, DDB2 binds DNA using its top face, and the positions occupied by CPSF-30 and PAS RNA in the quaternary complex has not been seen in the DDB1 complexes so far (SIAppendix, Fig. S9). The structural similarity between CPSF-160-WDR33 and DDB1-DDB2 suggests that they might be related evolutionarily. A system used for DNA damage repair might have been repur-posed for RNA processing (or vice versa).
While CPSF-160 is not directly involved in interactions with the PAS, the structure indicates that it has an essential role in this recognition, as a scaffold to recruit WDR33 and CPSF-30 and position them correctly for binding the PAS RNA Our EMSA data confirm that this preorganization of the binding site by the CPSF-160 scaffold is crucial for high-affinity PAS recognition.
Discussion
Our structure has illuminated the molecular mechanism for PAS recognition in pre-mRNA 3'-end processing, and it also provides a foundation for understanding and interpreting the biochemical data. Most importantly, earlier analyses have shown that AAUAAA is the predominant PAS among pre-mRNAs, with greater than 50% frequency (18, 19). The second most frequent PAS, AUUAAA, has a frequency of 16%, while all other PAS sequences have frequencies of less than 5%. Moreover, the U3 and A6 bases appear to be more conserved among the various PAS sequences. These observations are fully consistent with, and are explained by, our structure. For the AUUAAA hexamer, the recognition of the smaller U2 base could be mediated by a hydrogen bond between its carbonyl group on C4 and the main-chain amide of Lys78 in CPSF-30, which would only require a small rearrangement of the RNA. The AAUAAg mutation in
E1424  | www.pnas.org/cgi/doi/10.1073/pnas.1718723115
Sun et al.
CPSF-160-WDR33-CPSF-30
c ^ ^ mm
B
CPSF-160-WDR33-CPSF-30
> J <&      t? mm
AAUAAA C CPSF-160-WDR33
o   ^ J        ^ t? ^ MM
AAgAAA D MBP-CPSF-30
o ^        ^  1? *P MM
AAUAAA
AAUAAA
E      CPSF-160 + MBP-CPSF-30 F  CPSF-160-WDR33 + MBP-CPSF-30 mut
o   ^ «s> <£> ^   ^ £ MM o ^ ^>   ^   ^ MM
AAUAAA
AAUAAA
Fig. 5. CPSF-30 and WDR33 bind the PAS RNA synergistically. (/A) Interaction between the CPSF-160-WDR33-CPSF-30 ternary complex and the 17-mer PAS RNA with a 3' fluorescent label by EMSA. The position of the RNA alone is indicated with the thin arrow, and that of the stable complex with the thick arrow. The bracket marks a smeared band, indicating complexes that dissociated during the electrophoresis. (6) Interaction between the CPSF-160-WDR33-CPSF-30 ternary complex and a variant 17-mer PAS RNA, with AAgAAA hexamer. (C) Interaction between the CPSF-160-WDR33 binary complex and the PAS RNA. (D) Interaction between the MBP-CPSF-30 fusion protein and the PAS RNA. (F) Interaction between a mixture of CPSF-160 and MBP-CPSF-30 and the PAS RNA. (F) Interaction between a mixture of the CPSF-160-WDR33 binary complex and a mutant CPSF-30, MBP-CPSF-30(33-172), that cannot form the ternary complex and the PAS RNA. (6) Schematic drawing of the mammalian canonical 3'-end processing machinery. The organization of the CPSF-160-WDR33-CPSF-30-PAS RNA complex is based on the structure described here.
the human a2-globin gene causes a-thalassaemia (40) while the AAcAAA mutation in the human p-globin gene causes p-thal-assaemia (41). Both mutations disrupt the U3-A6 base pair and, thereby, 3'-end processing.
The structure also shows that ZF2-ZF3 of CPSF-30 is crucial for PAS recognition. This is supported by earlier data showing that deletion of either zinc finger abolished cross-linking to RNA and polyadenylation activity (16). Moreover, our structure explains how influenza virus NS1A protein can disrupt host pre-mRNA 3'-end processing by sequestering ZF2-ZF3 of CPSF-30 (42, 43). The structure of ZF2-ZF3 in the quaternary complex here is similar to that in the complex with NS1A while the bound position of NS1A clashes with the PAS RNA (Fig. AH). Therefore, CPSF-30 can no longer participate in the recognition of the AAUAAA PAS in the presence of NS1A.
The WD40 domain of WDR33 contacts the U3-A6 base pair, although there is no direct recognition of the bases by hydrogen bonds. In comparison, the WD40 domain of Gemin5 was reported earlier to specifically recognize the Sm site in pre-snRNAs (44, 45), and the RNA is bound mostly at the interface of the tandem WD40 domains (46-48). These results suggest diverse modes for WD40 domains to interact with RNA, in addition to their roles in protein-protein and protein-DNA interactions.
The PAS is a relatively short motif with only six nucleotides. The structure shows that the motif is divided into three pairs of two nucleotides each, A1-A2, A4-A5, and U3-A6, and each pair is recognized by a different protein component, which comes from two distinct proteins. Such a mode of interaction should enhance the specificity of the recognition, as each protein alone would have much lower affinity, which was demonstrated by our EMSA experiments. This binding mode also highlights the crucial importance of CPSF-160 as a scaffold, to preorganize CPSF-30 and WDR33 and allow them to bind synergistically to AAUAAA with high affinity. Therefore, the CPSF-160-WDR33-CPSF-30 ternary complex enables both high affinity and high selectivity in PAS recognition.
Many additional surface areas of CPSF-160 remain available in the complex, such as the bottom face of BP A, both faces of BPB, and the sides of all three propellers (Fig. IE), and CPSF-160 is likely to mediate interactions with other protein factors in the 3'-end processing machinery, for example CstF-77 (25, 49, 50). In fact, many of the equivalent surfaces in DDB1 have been found to interact with other proteins, and the protein bound between BPA and BPC does not have to contain a p-propeller either (30, 51) {SI Appendix, Fig. S9). Besides recruiting other proteins, it remains to be seen whether CPSF-160 could also interact with other regions of the pre-mRNA near the PAS.
Sun et al.
PNAS  |  Published online December 5, 2017  | E1425
The C-terminal segment of CPSF-30, including ZF4-ZF5 and the zinc knuckle, is disordered in the current structure. This segment may have other functions, such as interactions with Fipl. However, our construct of WDR33 contains only the N-terminal 572 residues, and our structure shows that the segment covering the N-terminal 420 residues is sufficient for recognizing the AAUAAA PAS and for interactions with CPSF-160 and CPSF-30. The role, if any, of the C-terminal segment of WDR33, including the collagen-like repeat, in 3'-end processing remains to be determined.
Overall, our studies have produced molecular insights into PAS recognition and the organization of the mPSF in the canonical 3'-end processing machinery (Fig. 5G). The hFipl subunit of mPSF is not involved in PAS recognition although it may interact with the pre-mRNA near the PAS (52, 53). hFipl also mediates the recruitment of the poly(A) polymerase (52, 54), enabling the polyadenylation reaction after cleavage. The mCF has strong interactions with the mPSF, forming CPSF (with sym-plekin), which together with CstF and other factors, defines the site of cleavage in the pre-mRNA. Further studies are needed to understand the molecular mechanism for these other steps in 3'-end processing.
Methods
Protein Expression and Purification. Human CPSF-160 and WDR33 (residues 1-572) were coexpressed in insect cells using Multibac technology (55) (Geneva Biotech). CPSF-160 and WDR33 were cloned into the pKL and pFL vectors, respectively, and a 6xHis tag was added to the N terminus of WDR33. Bacmids for expressing CPSF-160 and WDR33 were generated in DHIOEMBacY competent cells (Geneva Biotech) by transformation. Baculo-viruses were generated by transfecting bacmids into Sf9 cells using Cellfectin II (Thermo Fisher Scientific). P1 viruses were cultured at 27 °C for 5 d, and P2 viruses for large-scale infection were amplified from P1 viruses in 50 mL of Sf9 cells at 27 °C for 3 d. One liter of High5 cells (1.8 x 106 cells-mL-1) cultured in ESF 921 medium (Expression Systems) was infected with 15 mL of CPSF-160 P2 virus and 10 mL of WDR33 P2 virus at 27 °C with constant shaking. Cells were harvested after 48 h by centrifugation at 500 x g for 13 min.
For purification, the cell pellet was resuspended and lysed by sonication in 100 mL of buffer containing 25 mM Tris (pH 7.9), 300 mM NaCI, and one protease inhibitor mixture tablet (Sigma). The cell lysate was then centri-fuged at 24,000 x g for 45 min at 4 °C. The supernatant was incubated with nickel beads for 1 h at 4 °C. The beads were then washed four times with 50 bed volumes of wash buffer [25 mM Tris (pH 7.9), 300 mM NaCI, and 15 mM imidazole] and eluted with 25 mM Tris (pH 7.9), 150 mM NaCI, and 250 mM imidazole. The protein was further purified by chromatography using a HiTrap Q column (GE Healthcare) and a Hiload 16/60 Superdex 200 column (GE Healthcare). The CPSF-160-WDR33 complex was concentrated to 4 mg-tnL"1 in buffer containing 25 mM Tris (pH 7.9), 300 mM NaCI, and 5 mM DTT, and stored at -80 °C.
Human CPSF-30 (full length and residues 33-170) was cloned into the pRSFDuet vector (Novagen) and overexpressed in E. coli BL21 (DE3) Star cells. We used isoform 2 of CPSF-30, in which residues 191-215 are absent. A6xHis tag followed by MBP was added to the N terminus of CPSF-30, separated by a TEV protease cleavage site. Cell cultures were grown at 37 °C in LB-Agar (Sigma) containing 35 |igmL-1 kanamycin. When cell cultures reached an OD6oo of 0.7 ~ 0.8, they were supplemented with 0.2 mM ZnCI2 for 15 ~ 30 min, and protein expression was then induced with 0.15 mM isopropyl p-D-1-thiogalactopyranoside at 18 °C overnight. Cells were harvested by centrifugation at 1,000 x g for 15 min. The MBP-CPSF-30 fusion protein was purified following a similar protocol as that used for the CPSF-160-WDR33 complex.
CPSF-160-WDR33-CPSF-30-PAS RNA Quaternary Complex Formation. Purified CPSF-160-WDR33 complex and MBP-CPSF-30 were mixed at a molar ratio of 1:3 in the presence or absence of the RNA oligonucleotide AACCUC-CAAUAAACAAC (IDT). TEV protease was added at a ratio of 1:10 (wt/wt) with MBP-CPSF-30. The reaction mixture was incubated at room temperature for 2 h and then purified by gel filtration using a Superose 6 10/300 GL column (GE Healthcare), in a running buffer containing 25 mM Tris (pH 7.9), 350 mM NaCI, and 5 mM DTT. Samples from the quaternary complex fractions were used for EM studies. The presence of RNA in the complex was
confirmed with an A260/A280 ratio of 0.90, while the ratio was 0.64 if RNA was left out of the mixture.
EM Specimen Preparation and Data Collection. The homogeneity of the samples was first examined by negative-stain EM with 0.7% (wt/vol) uranyl formate as described (56). A Philips CM10 electron microscope operated at 100 kV was used to collect 54 images for the CPSF-160-WDR33 sample. The PAS RNA was added to the sample before preparing the EM grids, but no RNA density was observed in the eventual cryo-EM 3D reconstruction. The images were recorded at a defocus of -1.5 |im on an XR16L-ActiveVu charge-coupled device camera (AMT) at a nominal magnification of 52,000x (calibrated pixel size of 2.42 A on the specimen level) (SI Appendix, Fig. S1).
Before preparing grids for cryo-EM, the freshly purified protein samples were centrif uged at 13,000 x g for 2 min to remove protein aggregates, and the protein concentration was measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific). All specimens for cryo-EM were frozen with a Vitrobot Mark VI (FEI) set at 4 °C and 100% humidity. Cryo-EM imaging was performed in the Cryo-EM Resource Center at the Rockefeller University using SerialEM (57) or, when stated, in the Simons Electron Microscopy Center at the New York Structural Biology Center using Leginon (58).
The CPSF-160-WDR33 sample was concentrated to 1.8 mgmL-1 and CHAPSO (8 mM final concentration) was added to prevent the particles from adopting preferred orientations in the ice layer (59). A 3-|iL aliquot was applied to a glow-discharged Quantifoil 300 mesh 1.2/1.3 gold grid (Quan-tifoil). After 5 s, the grid was blotted for 3 s with a blot force setting of 0 and plunged into liquid ethane. Grids were screened with a Talos Arctica electron microscope, and 1,625 image stacks were collected on a 300-kV Titan Krios electron microscope (Thermo Fisher Scientific) at a defocus ranging from -1.3 to -2.7 |im. The images were recorded at a nominal magnification of 22,500x (calibrated pixel size of 1.3 A on the specimen level) with a K2 Summit camera in superresolution counting mode. Exposures of 10 s were dose-fractionated into 40 frames (250 ms per frame), with a dose rate of 8 electrons-pixel-1 s-1 (~1.18 electrons-A-2 per frame), resulting in a total dose of 47 electrons-A-2.
For the CPSF-160-WDR33-CPSF-30-PAS RNA sample, a 4-fiL aliquot at 0.18 mgmL-1 was applied to a glow-discharged Quantifoil 300 mesh 1.2/ 1.3 gold grid (Quantifoil). After 2 s, the grid was blotted for 4 s at a blot force setting of -2 and plunged into liquid ethane. Grids were screened with a Talos Arctica electron microscope, and 2,486 image stacks were collected on a Titan Krios electron microscope at the New York Structural Biology Center. The images were recorded with a K2 Summit camera in counting mode at a nominal magnification of 22,500x (calibrated pixel size of 1.07 A on the specimen level) and a defocus range from -1.2 to -2.5 |im. Exposures of 10 s were dose-fractionated into 40 frames (250 ms per frame), with a dose rate of 8 electronspixel-1s-1 (~1.75 electrons-A-2 per frame), resulting in a total dose of 70 electrons-A-2.
Image Processing. EMAN2 (60) was used to pick 27,300 particles of the negatively stained CPSF-160-WDR33 complex. The particles were windowed into 80 x 80-pixel boxes, which were then resized to 64 x 64 pixels. After centering, the particles were subjected to classification with the iterative stable alignment and clustering algorithm (61), specifying 100 images per group and a pixel error threshold of 0.7. Eight generations produced 412 averages (SI Appendix, Fig. S16). The class averages were used to calculate an initial model with the validation of individual parameter reproducibility algorithm implemented in SPARX (62) (SI Appendix, Fig. S1).
For the cryo-EM datasets, the image stacks were motion-corrected, dose-weighted, and binned over 2x2 pixels (binning was only performed for data collected in superresolution mode) in MotionCor2 (63). The CTF parameters were determined with CTFFIND4 (64).
For the CPSF-160-WDR33 dataset collected with the Titan Krios, 372,707 particles were automatically picked with Gautomatch (www.mrc-mb.cam.ac.uk/kzhang/Gautomatch/). After windowing the particles into 160 x 160-pixel boxes in RELION-2 (65), they were directly subjected to 3D classification into six classes, using the map obtained with the negative-stain EM dataset as initial model (2D classification in RELION-2 was performed but no particles were discarded). The orientation parameters of the particles in the largest class (containing 205,373 particles) were further refined, resulting in a final density map at 3.85-A resolution according to the gold-standard Fourier shell correlation (FSC) curve and a cutoff of 0.143 (SI Appendix, Fig. S2). Particle polishing improved the resolution to 3.78 A (SI Appendix, Fig. S3).
E1426  | www.pnas.org/cgi/doi/10.1073/pnas.1718723115
Sun et al.
For the CPSF-160-WDR33-CPSF-30-PAS RNA dataset collected with the Titan Krios, 1,144,122 particles were automatically picked with Gautomatch using as templates four class averages that were obtained from 2D classification of the cryo-EM dataset of the CPSF-160-WDR33 complex. The particles were windowed into 192 x 192-pixel boxes and subjected to 2D classification in RELION-2. Particles in classes showing clear structural features were combined (529,190 particles) and subjected to 3D classification into six classes using as initial model the density map obtained with vitrified CPSF-160-WDR33 complex filtered to 30-A resolution. Five classes produced maps with clear fine structure features, and the particles of these five classes were combined. Refinement yielded a density map at 3.36-A resolution, which showed high-resolution features of the CPSF-160-WDR33 region. Two of the six classes showed additional density, likely representing CPSF-30 and PAS RNA. These two classes were combined and subjected to another round of 3D classification into six classes. Five of the resulting classes that showed the additional density were combined (173,632 particles) and refinement yielded a map at 3.42-A resolution. To improve the density for the CPSF-30-PAS RNA region, additional refinement was performed using a soft mask containing BPC of CPSF-160, WDR33, CPSF-30, and PAS RNA. Particle polishing was performed but did not improve the map (SI Appendix, Fig. S2). The local resolution maps were calculated with the half maps in RELION-2.
Model Building and Refinement. We used predicted structures for CPSF-160 from l-TASSER (66) and WDR33 from Phyre2 (67), and the crystal structure of ZF2-ZF3 of CPSF-30 (43) (PDB ID code 2RHK) as starting models, and fitted them into the cryo-EM density map with Chimera (68) and Rosetta (69). All manual model building was performed with Coot (70). The atomic models were optimized by an iterative local rebuilding procedure in Rosetta using the map calculated with all of the data and then further refined by using phenix.real_space_refine (71) against half-map 1 from RELION-2. FSC curves were calculated between the refined models and half map 1 (work),
1. Proudfoot NJ (2011) Ending the message: Poly(A) signals then and now. Genes Dei/25: 1770-1782.
2. Yang Q, Doublie S (2011) Structural biology of poly(A) site definition. Wiley Interdiscip Rev RNA 2:732-747.
3. Xiang K, Tong L, Manley JL (2014) Delineating the structural blueprint of the pre-mRNA 3-end processing machinery. Mol Cell Biol 34:1894-1910.
4. Curinha A, Oliveira Braz S, Pereira-Castro I, Cruz A, Moreira A (2014) Implications of polyadenylation in health and disease. Nucleus 5:508-519.
5. Shi Y, Manley JL (2015) The end of the message: Multiple protein-RNA interactions define the mRNA polyadenylation site. Genes Dev 29:889-897.
6. Marzluff WF, Wagner EJ, Duronio RJ (2008) Metabolism and regulation of canonical histone mRNAs: Life without a poly(A) tail. Nat Rev Genet 9:843-854.
7. Romeo V, Schumperli D (2016) Cycling in the nucleus: Regulation of RNA 3' processing and nuclear organization of replication-dependent histone genes. Curr Opin Cell Biol 40:23-31.
8. Zhao J, Hyman L, Moore C (1999) Formation of mRNA 3' ends in eukaryotes: Mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol Mol Biol Rev 63:405^145.
9. Mandel CR, Bai Y, Tong L (2008) Protein factors in pre-mRNA 3'-end processing. Cell Mol Life Sci 65:1099-1122.
10. Shi Y, et al. (2009) Molecular architecture of the human pre-mRNA 3' processing complex. Mol Cell 33:365-376.
11. Bienroth S, Wahle E, Suter-Crazzolara C, Keller W (1991) Purification of the cleavage and polyadenylation factor involved in the 3'-processing of messenger RNA precursors. J Biol Chem 266:19768-19776.
12. Murthy KGK, Manley JL (1992) Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus. J Biol Chem 267:14804-14811.
13. Takagaki Y, Manley JL, MacDonald CC, Wilusz J, Shenk T (1990) A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs. Genes Dev 4: 2112-2120.
14. Gilmartin GM, Nevins JR (1991) Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA. Mol Cell Biol 11:2432-2438.
15. Mandel CR, et al. (2006) Polyadenylation factor CPSF-73 is the pre-mRNA 3-end-processing endonuclease. Nature 444:953-956.
16. Chan SL, et al. (2014) CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3' processing. Genes Dev 28:2370-2380.
17. Schonemann L, et al. (2014) Reconstitution of CPSF active in polyadenylation: Recognition of the polyadenylation signal by WDR33. Genes Dev 28:2381-2393.
18. Beaudoing E, Freier S, Wyatt JR, Claverie JM, Gautheret D (2000) Patterns of variant polyadenylation signal usage in human genes. Genome Res 10:1001-1010.
19. Tian B, Hu J, Zhang H, Lutz CS (2005) A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res 33:201-212.
20. Proudfoot NJ, Brownlee GG (1976) 3' non-coding region sequences in eukaryotic messenger RNA. Nature 263:211-214.
21. Elkon R, Ugalde AP, Agami R (2013) Alternative cleavage and polyadenylation: Extent, regulation and function. Wat Rev Genet 14:496-506.
half map 2 (free), and the combined map (SI Appendix, Fig. S3). The statistics from the structure determination is summarized in SI Appendix, Table S1.
Protein-RNA Binding Assays. Wild-type (AACCUCCAAUAAACAAC) and variant (AACCUCCAAgAAACAAC) 17-mer RNA oligos (both with 3'-end 6-FAM label; IDT) were dissolved in DEPC-treated water. The gel shift assays were performed with 0.2 |iM labeled wild-type or variant RNA and increasing concentrations of protein. The reactions were incubated at room temperature for 45 min in a 10-|jL volume containing 20 mM Tris (pH 7.9), 200 mM NaCI, 1 mM MgCI2, 1 mM DTT, 5% (vol/vol) glycerol, and 0.1 mg-mL"1 BSA. The samples were then supplemented with 1 |iL of 50% (vol/vol) glycerol and run on prechilled 0.6% (wt/vol) TAE (Tris, acetate, EDTA) agarose gels at 140 V for 30 min. The gels were visualized on a Typhoon FLA 7000 (GE Healthcare).
Sequence Alignment. Alignment of selected sequences of CPSF-30 and WDR33 homologs was produced with Clustal Omega (72), presented with ESPript (73), and modified manually to include additional information.
ACKNOWLEDGMENTS. We thank Mark Ebrahim and J. Sotiris for help with data collection at the Evelyn Gruss Lipper Cryo-Electron Microscopy Resource Center at The Rockefeller University; Ed Eng, Bill Rice, Laura Kim, and Bob Grassucci for help with data collection at the New York Structural Biology Center; Frank DiMaio for help with model building and refinement using Rosetta; and Kehui Xiang for initial studies on CPSF-160. This research is supported by NIH Grants R35GM118093 (to LT.) and R01GM090056 (to Y. Shi). Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by Simons Foundation Grant 349247; Empire State Development's Division of Science, Technology, and Innovation; and NIH National Institute of General Medical Sciences Grant GM103310 with additional support from the Agouron Institute Grant F00316 and NIH Grant S10 OD019994.
22. Gruber AR, Martin G, Keller W, Zavolan M (2014) Means to an end: Mechanisms of alternative polyadenylation of messenger RNA precursors. Wiley Interdiscip Rev RNA 5:183-196.
23. Tian B, Manley JL (2017) Alternative polyadenylation of mRNA precursors. Nat Rev Mol Cell Biol 18:18-30.
24. Sullivan KD, Steiniger M, Marzluff WF (2009) A core complex of CPSF73, CPSF100, and Symplekin may form two different cleavage factors for processing of poly(A) and histone mRNAs. Mol Cell 34:322-332.
25. Takagaki Y, Manley JL (2000) Complex protein interactions within the human polyadenylation machinery identify a novel component. Mol Cell Biol 20:1515-1525.
26. Kolev NG, Steitz JA (2005) Symplekin and multiple other polyadenylation factors participate in 3'-end maturation of histone mRNAs. Genes Dev 19:2583-2592.
27. Ghazy MA, He X, Singh BN, Hampsey M, Moore C (2009) The essential N terminus of the Pta1 scaffold protein is required for snoRNA transcription termination and Ssu72 function but is dispensable for pre-mRNA 3'-end processing. Mol Cell Biol 29: 2296-2307.
28. Xiang K, et al. (2010) Crystal structure of the human symplekin-Ssu72-CTD phos-phopeptide complex. Nature 467:729-733.
29. Jenny A, Keller W (1995) Cloning of cDNAs encoding the 160 kDa subunit of the bovine cleavage and polyadenylation specificity factor. Nucleic Acids Res 23: 2629-2635.
30. Li T, Chen X, Garbutt KC, Zhou P, Zheng N (2006) Structure of DDB1 in complex with a paramyxovirus V protein: Viral hijack of a propeller cluster in ubiquitin ligase. Cell 124:105-117.
31. Barabino SML, Hübner W, Jenny A, Minvielle-Sebastia L, Keller W (1997) The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins. Genes Dev 11:1703-1716.
32. De Guzman RN, et al. (1998) Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element. Science 279:384-388.
33. Rould MA, Perona JJ, Söll D, Steitz TA (1989) Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution. Science 246: 1135-1142.
34. Teplova M, et al. (2006) Structural basis for recognition and sequestration of UUU(OH) 3' temini of nascent RNA polymerase III transcripts by La, a rheumatic disease autoantigen. Mol Cell 21:75-85.
35. Aibara S, Gordon JMB, Riesterer AS, McLaughlin SH, Stewart M (2017) Structural basis for the dimerization of Nab2 generated by RNA binding provides insight into its contribution to both poly(A) tail length determination and transcript compaction in Saccharomyces cerevisiae. Nucleic Acids Res 45:1529-1538.
36. Teplova M, Patel DJ (2008) Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1. Nat Struct Mol Biol 15:1343-1351.
37. Park S, et al. (2017) Structural basis for interaction of the tandem zinc finger domains of human muscleblind with cognate RNA from human cardiac troponin T. Biochemistry 56:4154-4168.
38. Hudson BP, Martinez-Yamout MA, Dyson HJ, Wright PE (2004) Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d. Wat Struct Mol Biol 11: 257-264.
Sun et al.
PNAS  |  Published online December 5, 2017  | E1427
39. Scrima A, et al. (2008) Structural basis of UV DNA-damage recognition by the DDB1-DDB2 complex. Cell 135:1213-1223.
40. Higgs DR, et al. (1983) Alpha-thalassaemia caused by a polyadenylation signal mutation. Nature 306:398^00.
41. Orkin SH, Cheng TC, Antonarakis SE, Kazazian HH, Jr (1985) Thalassemia due to a mutation in the cleavage-polyadenylation signal of the human beta-globin gene. EMBO J 4:453^56.
42. Nemeroff ME, Barabino SML, Li Y, Keller W, Krug RM (1998) Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs. Mol Cell 1:991-1000.
43. Das K, et al. (2008) Structural basis for suppression of a host antiviral response by influenza A virus. Proc Natl Acad Sei USA 105:13093-13098.
44. Battle DJ, et al. (2006) The Gemin5 protein of the SMN complex identifies snRNAs. Mol Cell 23:273-279.
45. Lau CK, Bachorik JL, Dreyfuss G (2009) Gemin5-snRNA interaction reveals an RNA binding function for WD repeat domains. Nat Struct Mol Biol 16:486^491.
46. Xu C, et al. (2016) Structural insights into Gemin5-guided selection of pre-snRNAs for snRNP assembly. Genes Dev 30:2376-2390.
47. Jin W, et al. (2016) Structural basis for snRNA recognition by the double-WD40 repeat domain of Gemin5. Genes Dev 30:2391-2403.
48. Zuo Y, Deutscher MP (2001) Exoribonuclease superfamilies: Structural analysis and phylogenetic distribution. Nucleic Acids Res 29:1017-1026.
49. Murthy KGK, Manley JL (1995) The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation. Genes Dev 9:2672-2683.
50. Bai Y, et al. (2007) Crystal structure of murine CstF-77: Dimeric association and implications for polyadenylation of mRNA precursors. Mol Cell 25:863-875.
51. Angers S, et al. (2006) Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 443:590-593.
52. Kaufmann I, Martin G, Friedlein A, Langen H, Keller W (2004) Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase. EMBO J 23:616-626.
53. Martin G, Gruber AR, Keller W, Zavolan M (2012) Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length. Cell Rep 1:753-763.
54. Meinke G, et al. (2008) Structure of yeast poly(A) polymerase in complex with a peptide from Fip1, an intrinsically disordered protein. Biochemistry 47:6859-6869.
55. Sari D, et al. (2016) The MultiBac baculovirus/insect cell expression vector system for producing complex protein biologies. Adv Exp Med Biol 896:199-215.
56. Ohi M, Li Y, Cheng Y, Walz T (2004) Negative staining and image classification-Powerful tools in modern electron microscopy. Biol Proced Online 6:23-34.
57. Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36-51.
58. Suloway C, et al. (2005) Automated molecular microscopy: The new Leginon system. J Struct Biol 151:41-60.
59. Kang JY, et al. (2017) Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex. Elife 6:e25478.
60. Tang G, et al. (2007) EMAN2: An extensible image processing suite for electron microscopy. J Struct Biol 157:38-46.
61. Yang Z, Fang J, Chittuluru J, Asturias FJ, Penczek PA (2012) Iterative stable alignment and clustering of 2D transmission electron microscope images. Structure 20:237-247.
62. Hohn M, et al. (2007) SPARX, a new environment for Cryo-EM image processing. J Struct Biol 157:47-55.
63. Zheng SQ, et al. (2017) MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 14:331-332.
64. Rohou A, Grigorieff N (2015) CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J Struct Biol 192:216-221.
65. Kimanius D, Forsberg BO, Scheres SH, Lindahl E (2016) Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. Elife 5:e18722.
66. Roy A, Kucukural A, Zhang Y (2010) l-TASSER: A unified platform for automated protein structure and function prediction. Nat Protoc 5:725-738.
67. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845-858.
68. Pettersen EF, et al. (2004) UCSF Chimera—A visualization system for exploratory research and analysis. J Comput Chem 25:1605-1612.
69. Wang RY, et al. (2016) Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. Elife 5:e17219.
70. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126-2132.
71. Adams PD, et al. (2002) PHENIX: Building new software for automated crystallo-graphic structure determination. Acta Crystallogr D Biol Crystallogr 58:1948-1954.
72. Sievers F, et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539.
73. Gouet P, Courcelle E, Stuart Dl, Metoz F (1999) ESPript: Analysis of multiple sequence alignments in PostScript. Bioinformatics 15:305-308.
E1428   |  www.pnas.org/cgi/doi/10.1073/pnas.1718723115
Sun et al.