Staufen2 functions in Staufen1-mediated mRNA decay INAUGURAL ARTICLE by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity

Eonyoung Parka,b, Michael L. Gleghorna,b, and Lynne E. Maquata,b,1

aDepartment of Biochemistry and Biophysics, School of Medicine and Dentistry, and bCenter for RNA Biology, University of Rochester, Rochester, NY 14642

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2011.

Edited by Michael R. Botchan, University of California, Berkeley, CA, and approved November 16, 2012 (received for review August 3, 2012)

Staufen (STAU)1-mediated mRNA decay (SMD) is a posttranscrip- harbor a STAU1-binding site (SBS) downstream of their normal tional regulatory mechanism in mammals that degrades mRNAs termination codon in a pathway called STAU1-mediated mRNA harboring a STAU1-binding site (SBS) in their 3′-untranslated regions decay or SMD (13, 14), and work published by others indicates (3′ UTRs). We show that SMD involves not only STAU1 but also its that SMD does not involve STAU2 (3, 15). paralog STAU2. STAU2, like STAU1, is a double-stranded RNA-binding According to our current model for SMD, when translation that interacts directly with the ATP-dependent RNA helicase terminates upstream of an SBS, recruitment of the nonsense-me- diated mRNA decay (NMD) factor UPF1 to SBS-bound STAU1 up-frameshift 1 (UPF1) to reduce the half-life of SMD targets that form fl an SBS by either intramolecular or intermolecular base-pairing. Com- triggers mRNA decay. SMD in uences a number of cellular pro- pared with STAU1, STAU2 binds ∼10-foldmoreUPF1and∼two- to cesses, including the differentiation of mouse C2C12 myoblasts to myotubes (16), the motility of human HaCaT keratinocytes (17), fivefold more of those SBS-containing mRNAs that were tested, and it and the differentiation of mouse 3T3-L1 preadipocytes to adipo- comparably promotes UPF1 helicase activity, which is critical for SMD. cytes (18). Thus, SMD may explain the essential role of STAU1 in STAU1- or STAU2-mediated augmentation of UPF1 helicase activity is embryonic stem-cell differentiation (19). not accompanied by enhanced ATP hydrolysis but does depend on ATP

We report here that SMD is triggered not only by STAU1 but BIOCHEMISTRY binding and a basal level of UPF1 ATPase activity. Studies of STAU2 also by STAU2, each of which can self-associate as well as as- demonstrate it changes the conformation of RNA-bound UPF1. These sociate with one another. Our findings that SMD requires the findings, and evidence for STAU1−STAU1, STAU2−STAU2, and ATP-dependent helicase activity of UPF1, each STAU paralog STAU1−STAU2 formation in vitro and in cells, are consistent with promotes UPF1 helicase activity without promoting UPF1 ATP results from tethering assays: the decrease in mRNA abundance hydrolytic activity, and STAU2 increases the RNA footprint of brought about by tethering siRNA-resistant STAU2 or STAU1 to an UPF1 allow us to expand on the existing model for SMD. We mRNA 3′ UTR is inhibited by downregulating the abundance of cel- propose that STAU paralog-binding to UPF1 induces a change lular STAU2, STAU1, or UPF1. It follows that the efficiency of SMD in in UPF1 conformation that resembles its conformation during different cell types reflects the cumulative abundance of STAU1 and intermediate or product steps of ATP hydrolysis and enhances STAU2. We propose that STAU paralogs contribute to SMD by “greas- the amount of unwinding per ATP molecule hydrolyzed. ing the wheels” of RNA-bound UPF1 so as to enhance its unwinding capacity per molecule of ATP hydrolyzed. Results STAU2 Functions in SMD. Human embryonic kidney (HEK) 293T protein–protein interactions | protein-RNA interactions and HeLa cells express multiple isoforms of STAU2 and STAU1 (1, 13, 20) (Fig. 1A and Fig. S1A). STAU2 and STAU1 are ∼50% identical (20). dsRBDs 3 and 4 have retained the ability to bind ammalian cells contain two Staufen (STAU) paralogs, dsRNA and are 78% and 81% identical, respectively (2). MSTAU1 and STAU2. Each derives from a separate dsRBD2, which is 48% identical between the two paralogs, and that produces multiple protein isoforms via alternative pre-mRNA dsRBD5, the full-length of which is unique to STAU1 (21) (Fig. splicing and/or . Every STAU1 and STAU2 1A), have been implicated in the formation of STAU1 homo- isoform contains multiple conserved dsRNA-binding domains dimers and homomultimers in complex with RNA (22). (RBDs), of which dsRBD3 and dsRBD4 constitute the major if To assay for STAU2 function in SMD, STAU2 and, for com- not sole active dsRNA-binding sites (1, 2). parison, STAU1 were down-regulated to the same extent in HeLa Although STAU1 and probably STAU2 are expressed ubiq- cells, where their abundance is comparable (Fig. S1 B and C). uitously, STAU2 is most abundant in heart and brain (1, 3). Both Subsequently, the levels of SMD reporters (normalized to the level paralogs are present in ribonucleoprotein particles (RNPs) within of a reference mRNA to control for variations in cell transfection) neuronal cell bodies and dendrites (1, 4–7). Both are also in- and a cellular SMD target (normalized to the level of its pre- volved in the -dependent transport of to mRNA to control for effects on transcription) were quantitated. dendrites of polarized neurons (5, 8), presumably via the tubulin- Thus, HeLa cells were transiently transfected with the phCMV- binding domain (2). Moreover, both paralogs play a crucial role in MUP [which produces major urinary protein (MUP) mRNA the formation and maintenance of the dendritic spines of hippo- driven by a cytomegalovirus (CMV) promoter] reference plasmid campal neurons and are required for synaptic plasticity and memory (7, 9–11), although the detection of paralog-specific RNA granules in the distal dendrites of rat hippocampal neurons Author contributions: E.P., M.L.G., and L.E.M. designed research; E.P. and M.L.G. per- has been used to argue for paralog-distinct functions (1). Addi- formed research; E.P., M.L.G., and L.E.M. analyzed data; and E.P., M.L.G., and L.E.M. wrote tional support for paralog-specific roles derives from the dem- the paper. onstration using rat hippocampal slice cultures that STAU1 but The authors declare no conflict of interest. not STAU2 functions in the late phase of forskolin-induced long- This article is a PNAS Direct Submission. – term potentiation (10 12), whereas STAU2 but not STAU1 is 1To whom correspondence should be addressed. E-mail: [email protected]. involved in metabotropic glutamate receptor-mediated long- edu. term depression (12). As another example of apparent paralog This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. specificity, we have shown that STAU1 degrades mRNAs that 1073/pnas.1213508110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1213508110 PNAS Early Edition | 1of8 Downloaded by guest on September 29, 2021 mentary Alu elements (17). One day later, cells were transiently A RBD2 RBD3 RBD4 TBD RBD5 STAU163 1 577 transfected with Control, STAU1, or STAU2 siRNA. STAU155 1 496 Western blotting (WB) demonstrated that STAU1 and STAU2 RBD1 fi STAU262 1 570 siRNAs speci cally reduced the levels of their target isoforms STAU259 1 538 to <10% the levels in Control siRNA-treated cells (Fig. 1C). STAU252 1 479 RT-PCR revealed that STAU1 and STAU2 siRNAs augmented B FLUC the level of each SMD reporter mRNA ∼two- to threefold, in- pcFLUC GAP43 3’UTR dicating that each paralog functions in SMD to a comparable extent SERPINE1 3’UTR D – c-JUN SBS [Fig. 1 ; corroborating RT quantitative PCR (qPCR) data in ARF1 SBS Fig. S1D and data obtained using STAU2(A) or STAU2(B) siRNA FLJ21870 3’UTR in Fig. S1 E and F]. Consistent with STAU2 function in SMD, C STAU2 siRNA, like STAU1 siRNA, up-regulated the level of en- siRNA dogenous GAP43 mRNA (Fig. 1D and Fig. S1F) and reduced the STAU2 Control STAU1 STAU163 (WB: -STAU1) half-life of mRNA harboring a 3′ UTR SBS (Fig. S1 G–I). STAU155 STAU262 STAU259 (WB: -STAU2) STAU252 STAU2, Like STAU1, Interacts Directly with UPF1. To begin to assess Calnexin (WB: -Calnexin) STAU2 isoforms for function in SMD, extracts of HeLa cells that stably express FLAG-UPF1 at ≤twofold the level of cellular D siRNA STAU1 Control STAU2 UPF1 (23) were immunoprecipitated in the presence or absence FLUC-GAP43 3’UTR mRNA of RNase A using anti-FLAG or, as a control for nonspecific 36 FLUC-GAP43 3’UTR immunoprecipitation (IP), mouse (m)IgG. Approximately 10% mRNA / MUP mRNA (%) 100 246 16 220 of each isoform of STAU1 or STAU2 coimmunoprecipitated FLUC-SERPINE1 3’UTR mRNA with FLAG-UPF1 (Fig. 2A). Furthermore, compared with the FLUC-SERPINE1 3’UTR mRNA / MUP mRNA (%) STAU1 isoforms (see also ref. 18), the co-IP efficiency of each 268 20 100 304 2 FLUC-c-JUN SBS mRNA FLUC-c-JUN SBS mRNA / MUP mRNA (%) 184 24 100 223 34 +- - + RNase A FLUC-ARF1 SBS mRNA A FLUC-ARF1 SBS mRNA / IP -FLAG MUP mRNA (%) -FLAG mIgG -- mIgG 168 2 100 224 17 FLUC-FLJ21870 3’UTR mRNA FLAG-UPF1 (WB: -FLAG) 63 STAU1 (WB: -STAU1) FLUC-FLJ21870 3’UTR * * * STAU155 mRNA / MUP mRNA (%) 63 100100 STAU1 (%) 289 54 289 67 100 12 2 GAP43 pre-mRNA STAU155 (%)

GAP43 mRNA 92 STAU262 59 GAP43 mRNA / STAU2 (WB: -STAU2) * * * STAU252 GAP43 pre-mRNA (%) STAU262 224 12 100 230 6 STAU259 Darker exposure MUP mRNA 62

100 STAU2 (%)

GAPDH mRNA 84 5

59

100 STAU2 (%) Fig. 1. siRNA-mediated down-regulation of STAU2 inhibits SMD. (A)Diagrams 90 3 2 52 of human STAU1 and STAU2 isoforms, where boxes represent functional and/or 100 STAU2 (%) 86 structural domains. 1, N-terminal amino acid; TBD, tubulin-binding domain UPF2 (WB: -UPF2) (where the hatched STAU2 TBD is ∼18% identical to the STAU1 TBD). (B)Partial

100 UPF2 (%) 6 diagrams of pcFLUC SMD-reporter plasmids. (C and D)HeLacells(4× 10 per 48 6 μ PABPC1 (WB: -PABPC1) 100-mm dish) were transiently transfected with 1.5 g of each pcFLUC SMD- Calnexin (WB: -Calnexin) reporter plasmid and 1 μg of the phCMV-MUP reference plasmid. Cells were -+ RNase A retransfected 1 d later with 100 nM of Control siRNA, STAU1 siRNA, or STAU2 SMG7 mRNA siRNA and harvested 2 d later. (C) WB, where Calnexin controlled for variations in protein loading. The left-most four lanes show serial threefold dilutions of B Before After FLAG-UPF1 pull-down 55 55 62 62 59 protein. Arrows pointing right denote STAU isoforms, and unidentified bands 59 do not interfere with the analyses. (D) RT-PCR, where the level of each FLUC GST- STAU1 STAU1 STAU2 STAU2 PABPC1 STAU2 PABPC1 STAU2 mRNA was normalized to the level of MUP mRNA, the level of cellular GAP43 FLAG-UPF1 (WB: -FLAG) GST-PABPC1 mRNA was normalized to the level of its pre-mRNA, and normalized levels in GST-STAU262 (WB: GST-STAU259 -GST) the presence of Control siRNA were defined as 100. All results are representa- GST-STAU155

100 After / before pull-down (%) 81

tive of at least three independently performed experiments. 77 4

Fig. 2. Compared with STAU1 isoforms, STAU2 isoforms interact more effi- × 7 and a mixture of the five pcFLUC SMD-reporter plasmids, ciently with UPF1. (A) HeLa cells stably expressing FLAG-UPF1 (8 10 per 150- mm dish) were lysed and analyzed before (−) or after IP using anti(α)-FLAG or, which encode firefly luciferase (FLUC) mRNAs and vary in − + ′ B ′ as a negative control, mouse (m)IgG in the absence ( )orpresence( )ofRNase their 3 UTRs (Fig. 1 ) (13, 14, 17). The 3 UTRs consist of the A. WB was performed, where the left-most four lanes show threefold dilutions growth-associated protein 43 (GAP43) 3′ UTR, the serpin pepti- of protein before IP. Arrows pointing right and red dots denote STAU isoforms, dase inhibitor, clade E (nexin, plasminogen activator inhibitor and the asterisks specify unidentified . The level of each coimmuno- type 1), member 1 (SERPINE1) 3′ UTR, the v-jun avian sarcoma precipitated STAU protein in the absence of RNase A was defined as 100. (A, virus 17 oncogene homolog (c-JUN) SBS (nucleotides 485–675 Lower) SMG7 mRNA was analyzed using RT-PCR before IP to demonstrate that of the c-JUN 3′ UTR), the ADP-ribosylation factor 1 (ARF1) the RNase A digestion was complete. The left-most six lanes represent twofold – ′ dilutions of RNA. (B)GST-STAU155,GST-STAU262,GST-STAU259,orGST-PABPC1 SBS (nucleotides 1 300 of the ARF1 3 UTR), or the ankyrin- fi repeat domain 57 (FLJ21870, also called ANKRD57) 3′ UTR. puri ed from E. coli (100 nM) was incubated with baculovirus-produced and purified FLAG-UPF1 (200 nM), subjected to FLAG-UPF1 pull-down, and analyzed The ARF1 SBS and probably the GAP43 and c-JUN SBSs are using WB. Protein levels after pull-down were normalized to the level before formed by intramolecular base-pairing, whereas the SERPINE1 pull-down, and the normalized level of GST-STAU262 was defined as 100. The and FLJ21870 SBSs are formed by intermolecular base-pairing left-most four lanes show threefold dilutions of protein. Red arrowheads de- with a long-noncoding RNA (1/2-sbsRNA1) via partially comple- note the GST-STAU protein under analysis (Fig. S2B).

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1213508110 Park et al. Downloaded by guest on September 29, 2021 STAU2 isoform was ∼10-fold more resistant to RNase A (Fig. A Before After GST-STAU155 pull-down +---+--- 55 2A). As indications that RNase A treatment was successful, the co- HIS-STAU1 INAUGURAL ARTICLE -+---+-- STAU262-HIS IP of UPF2 with FLAG-UPF1 was partially sensitive to RNase A --+---+- STAU259-HIS ---+---+ BSA (Fig. 2A) (24), the co-IP of poly(A)-binding protein C1 (PABPC1) GST-STAU155 STAU262-HIS or BSA was completely sensitive to RNase A (Fig. 2A) (24), and cellular STAU259-HIS HIS-STAU155 suppressor with morphogenetic effect on genitalia protein 7 (SMG7) mRNA was detectable in the absence but not the presence of B Before After STAU262-HIS pull-down A +---+--- GST-STAU155 RNase A (Fig. 2 ). IPs of cellular UPF1 using lysates from -+---+-- STAU262-GST --+---+- STAU259-GST formaldehyde cross-linked human neuroblastoma SK-N-MC cells ---+---+ GST-PABPC1 demonstrated that STAU1 and STAU2 isoforms also coimmuno- STAU262-HIS GST-PABPC1 STAU262-GST (WB: precipitate with cellular UPF1, indicating that the interactions oc- STAU259-GST -GST) cur within cells rather than as an artifact after cell lysis (Fig. S2A). GST-STAU155 To examine whether the interaction of STAU2 with UPF1 is C Before After STAU259-HIS pull-down direct, and to determine whether the higher association of UPF1 +---+--- GST-STAU155 -+---+-- STAU262-GST with STAU2 relative to STAU1 is recapitulated in vitro, GST- --+---+- STAU259-GST 55 62 59 ---+---+ GST-PABPC1 STAU1 (13), GST-STAU2 , GST-STAU2 or, as a negative STAU259-HIS fi Escher- GST-PABPC1 control, GST-PABPC1 was produced and puri ed from STAU262-GST (WB: STAU259-GST -GST) ichia coli, mixed with baculovirus-produced and purified FLAG- GST-STAU155 UPF1 (25), and subjected to FLAG-UPF1 pull-down. Of the +- - + RNase A STAU2 isoforms, we focused on STAU262 and STAU259 because D IP their binding to mRNA has been characterized using a genome- -HA -HA rIgG -- rIgG 55 wide approach (20). FLAG-UPF1 pulled down ∼10-fold more STAU1 -HA3 (WB: -HA) 55 63 (WB: 62 59 55 STAU1 -HA3+STAU1 GST-STAU2 and GST-STAU2 compared with GST-STAU1 * * * STAU155 - 55 STAU1) B B 83 STAU1 (%) (Fig. 2 and Fig. S2 ). As expected, FLAG-UPF1 failed to pull 100 STAU262 B STAU259 (WB: -STAU2) down GST-PABPC1 (Fig. 2 ). These results are consistent with * STAU252 fi STAU262 (%)

the nding that the co-IP of STAU2 with FLAG-UPF1 is less 100 102 59 sensitive to RNase A than the co-IP of STAU1 with FLAG-UPF1. 96 STAU2 (%) 100 STAU252 (%) 100 117 Calnexin (WB: -Calnexin) STAU2 and STAU1 Paralogs Self-Associate and Associate with One BIOCHEMISTRY

Another. STAU1 has been found to dimerize if not multimerize -+ RNase A with itself in vitro and in cells (22). To better define STAU2 in- GAPDH mRNA E. coli teractions in vitro, pull-downs using -produced proteins - -HA IP C 55 (Fig. S2 ) were performed. GST-STAU1 pulled down not only E ---+-+ 55 62 59 RNase A +-++-- pCI-neo-HA3 HIS-STAU1 but also STAU2 -HIS and STAU2 -HIS with 62 -+--++ pCI-neo-STAU2 -HA3 fi 62 comparable ef ciencies and, as expected, did not pull down BSA STAU2 -HA3 (WB: -HA) 62 59 59 (Fig. 3A). Furthermore, STAU2 -HIS and STAU2 -HIS pulled STAU2 (WB: -STAU2) 55 62 STAU252 59 down a comparable amount of GST-STAU1 , STAU2 -GST, 90 STAU2 (%) 59 100 or STAU2 -GST and, as expected, did not pull down GST- STAU252 (%) 100 110 PABPC1 (Fig. 3 B and C) (26). These findings indicate that the PLC 1 (WB: -PLC 1) efficiencies with which each STAU2 isoform interacts with itself, 55 -+-+ RNase A with one another, or with STAU1 are indistinguishable in vitro GAPDH mRNA (Fig. S2D). Fig. 3. STAU262 and STAU259 self-associate and associate with one another To assay for STAU1 and STAU2 interactions in vivo, 55 55 HEK293T cells were transiently transfected with a STAU155- and with STAU1 .(A)GST-STAU1 (100 nM) was incubated with 100 nM of HIS-STAU155,STAU262-HIS, STAU259-HIS or, as a negative control, BSA, sub- HA3 expression vector (13, 14; where it was called pSTAU1-HA) 55 55 jected to GST-STAU1 pull-down, resolved in SDS-polyacrylamide, and stained to test for the ability of STAU1 -HA3 to interact with cellular 55 using SYPRO Ruby. The left-most four lanes are threefold dilutions of GST- STAU1 and STAU2. STAU1 -HA3 indeed coimmunoprecipi- 55 55 62 59 52 STAU1 and BSA. (B and C) WB before and after HIS pull-down of 100 nM of tated with cellular STAU1 , STAU2 , STAU2 , and STAU2 STAU262-HIS (B)orSTAU259-HIS (C) that was incubated with 100 nM of GST- in the absence or presence of RNase A (Fig. 3D; it was not pos- 55 62 59 63 STAU1 ,STAU2 -GST, STAU2 -GST or, as a negative control, GST-PABPC1. (D) sible to assay cellular STAU1 because it comigrates with HEK293T cells (4 × 107 per 150-mm dish) were transiently transfected with 10 μg 55 55 STAU1 -HA3). Control experiments demonstrated that none of of pSTAU1 -HA3, lysed, and analyzed before (−) or after IP using anti(α)-HA or, the STAU isoforms was immunoprecipitated using rat (r)IgG, as a negative control, rat (r)IgG in the absence (−)orpresence(+)ofRNaseA. Calnexin failed to coimmunoprecipitate under any circumstance, The level of each coimmunoprecipitated STAU protein in the absence of RNase and cellular GAPDH mRNA was detectable in the absence but Awasdefined as 100. Arrows pointing left and red dots denote STAU iso- not the presence of RNase A (Fig. 3D). In related experiments, forms, and the asterisks specify unidentified proteins. Calnexin controlled 62 fi HEK293T cells were transiently transfected with a STAU2 -HA3 for variations in protein loading before IP and demonstrates the speci city 62 of the IP. (D, Lower) GAPDH mRNA was analyzed using RT-PCR before IP to expression vector. STAU2 -HA3 also coimmunoprecipitated with 59 52 demonstrate that the RNase A digestion was complete. The six left-most cellular STAU2 and STAU2 in the absence or presence of 7 RNase A (Fig. 3E; it was not possible to assay cellular STAU262 lanes represent twofold serial dilutions of RNA. (E) HEK293T cells (4 × 10 per 62 150-mm dish) were transiently transfected with 5 μg of pCI-neo-HA3 or pCI- because it comigrates with STAU2 -HA3). Control experiments 62 α showed that none of the STAU isoforms was immunoprecipitated neo-STAU2 -HA3, lysed, and analyzed before or after IP using -HA in the in mock transfections involving an HA expression vector, PLCγ1 absence or presence of RNase A. The level of each coimmunoprecipitated 3 STAU1 isoform in the absence of RNase A was defined as 100. Notations, failed to coimmunoprecipitate under any circumstance, and cel- serial dilutions, and GAPDH mRNA analyses were as in D. lular GAPDH mRNA was detectable in the absence but not the presence of RNase A (Fig. 3E).

STAU2, Like STAU1, Coimmunoprecipitates with SBSs. STAU1 and next determined whether STAU2 coimmunoprecipitates with STAU2 have been reported to bind distinct but overlapping RNA sequences that are known to bind STAU1 and trigger subsets of mRNAs in HEK293T cells (20). Considering our SMD (13, 14, 16). findings that STAU2 and STAU1 interact directly (Fig. 3) and HeLa cells were transiently transfected with a pCI-neo- 55 62 59 all STAU2 isoforms associate with polysomes (Fig. S2E), we STAU1 -HA3, pCI-neo-STAU2 -HA3, or pCI-neo-STAU2 -

Park et al. PNAS Early Edition | 3of8 Downloaded by guest on September 29, 2021 HA3 effector plasmid together with four of the pcFLUC SMD- A MS2bs reporter plasmids (Fig. 1B). Binding studies were performed x 8 using HeLa cells because, as noted above, they contain compa- pcFLUC-MS2bs FLUC FLUC rable amounts of endogenous STAU1 and STAU2 paralogs (Fig. pcFLUC 55 62 S1 B and C), and comparable levels of STAU1 -HA3, STAU2 - pcFLUC- 59 B pcFLUC MS2bs HA3, and STAU2 -HA3 can be expressed. The pcFLUC SMD- 55 55 62 59 reporter plasmids served as positive controls for STAU155 binding. 62 59 pMS2-HA- Two days after transfection, cells were exposed to formaldehyde, STAU1 STAU1 STAU2 STAU2 - STAU2 STAU2 - MS2-HA-STAU262 and lysates were immunoprecipitated using anti-HA. 59 (WB: MS2-HA-STAU2 -HA) STAU155-HA ,STAU262-HA , and STAU259-HA were ex- MS2-HA-STAU155 3 3 3 PLC 1 (WB: -PLC 1) pressed at comparable levels before IP and immunoprecipitated 62 FLUC or with comparable efficiencies (Fig. 4A). STAU2 -HA3 and FLUC-MS2bs mRNA 59 MUP mRNA 7 8 STAU2 -HA3 coimmunoprecipitate with FLUC SMD-reporter 4 ∼ fi fi 55 mRNAs two- to vefold more ef ciently than does STAU1 - FLUC or 102 111 129 100 FLUC-MS2bs mRNA (%) HA3 (Fig. 4 B and C). Because the efficiency of paralog self-as- 13 2 10 100 50 sociation and association with one another is comparable, and the 50 42 55 co-IP of STAU1 -HA3 with the reporter mRNAs reflects binding to the SBSs, we conclude that STAU262 and STAU259 may bind C Control UPF1 siRNA 59 55 62 other if not all SBSs more efficiently than does STAU155. 55 62 59 pMS2-HA- STAU2 STAU1 STAU2 - STAU1 STAU2 - STAU2 MS2-HA-STAU262 (WB: Tethering siRNA-Resistant STAU2 or STAU1 Downstream of a MS2-HA-STAU259 MS2-HA-STAU155 -HA) Termination Codon Reduces mRNA Abundance in a Way That Is UPF1 (WB: -UPF1) Inhibited When Cellular STAU1, STAU2, or UPF1 Is Down-Regulated. PLC 1 (WB: -PLC 1) Tethering human STAU1 sufficiently downstream of the termi- D 140 nation codon of a reporter mRNA triggers SMD of that mRNA 120 depending on UPF1 and translation (13, 25). Given our findings 100 that down-regulating either STAU1 or STAU2 inhibits SMD 80 62 59 60 (Fig. 1 and Fig. S1), and STAU2 and STAU2 bind UPF1, 40 20 STAU1, and SBSs (Figs. 2–4), tethering STAU2 isoforms should RLUC mRNA (%)

FLUC-MS2bs mRNA / 0 also trigger SMD in a pathway that involves cellular UPF1 and both cellular STAU paralogs. To test this hypothesis because the E Control STAU1STAU2 siRNA 62R 59R 62R 59R literature indicates otherwise (3, 15), HeLa cells were transiently 55R 55R 55R 62R 59R pMS2-HA- STAU2 STAU2 STAU2 STAU2 - STAU1 - STAU1 - STAU1 STAU2 STAU2 MS2-HA-STAU262R 59R (WB: MS2-HA-STAU2 -HA) MS2-HA-STAU155R - -HA IP 63 STAU1 (WB: -STAU1) A STAU155 +---+--- pCI-neo-HA3 -+---+-- 55 STAU262 pCI-neo-STAU1 -HA3 59 --+---+- 62 STAU2 (WB: -STAU2) pCI-neo-STAU2 -HA3 52 ---+---+ 59 STAU2 pCI-neo-STAU2 -HA3 PLC 1 (WB: -PLC 1) STAU262-HA 59 3 STAU2 -HA3 (WB: -HA)

55 / FLUC-MS2bs mRNA STAU1 -HA3 F PLC 1 (WB: -PLC 1) 140 RLUC mRNA (%) 120 100

IP) 800 FLUC-GAP43 80

B - 3’UTR mRNA 60 600 IP / FLUC-SERPINE1 40 + 400 3’UTR mRNA 20 FLUC-c-JUN 0 200 SBS mRNA FLUC-FLJ21870 55 62 0 3’UTR mRNA Fig. 5. Evidence that tethering MS2-HA-STAU1 ,MS2-HA-STAU2 ,orMS2- % of ( mRNA STAU155 STAU262 STAU259 pCI-neo- HA-STAU259 to the FLUC-MS2bs mRNA 3′ UTR triggers FLUC-MS2bs mRNA SMD -HA3 -HA3 -HA3 in a mechanism that depends on the cellular STAU paralogs and cellular UPF1. C -HA IP (A) Schematic representations of pcFLUC and pcFLUC-MS2bs, where × 8specifies +--- pCI-neo-HA3 eight tandem repeats of the MS2 coat protein-binding site (MS2bs). (B) HeLa 55 -+-- pCI-neo-STAU1 -HA3 5 --+- 62 cells (2.5 × 10 per well of a six-well plate) were cotransfected with 0.2 μgof pCI-neo-STAU2 -HA3 ---+ pCI-neo-STAU259-HA 3 pcFLUC or pcFLUC-MS2bs, 0.1 μgofphCMV-MUP,and1μg of the specified E.coli LacZ mRNA pMS2-HA-STAU effector plasmid. Two days after transfection, protein and RNA 62 59 fi α Fig. 4. STAU2 -HA3 and STAU2 -HA3 coimmunoprecipitate with known were puri ed. WB using anti( )-HA demonstrated comparable levels of effector 55 SBS-containing targets more efficiently than does STAU1 -HA3. HeLa cells protein expression. (B, Lower) Using RT-PCR, normalized levels of FLUC or FLUC- (3 × 107 per 150-mm dish) were transiently transfected with 2 μg of each MS2bs mRNA in the presence of the specified effector protein were calculated 55 pcFLUC SMD-reporter plasmid, and 5 μg of pCI-neo-HA3, pCI-neo-STAU1 - as a percentage of the normalized level of FLUC or FLUC-MS2bs mRNA in the 62 59 fi × 5 HA3, pCI-neo-STAU2 -HA3, or pCI-neo-STAU2 -HA3 effector plasmid. Two presence of pMS2-HA, which was de ned as 100. (C and D) HeLa cells (2.5 10 days later, lysates from formaldehyde-treated cells were immunoprecipi- per well) were transiently transfected with 0.1 μg of pcFLUC-MS2bs, 0.1 μgof tated using anti-HA, and cross-links were reversed. (A) WB of cell lysates a plasmid that produces RLUC protein (pRLUC), which encodes renilla luciferase, after IP relative to before (−) IP using the specified antibody (α). PLCγ1 and 1 μg of the specified effector plasmid. One day later, cells were transfected controlled for variations in protein loading before IP and demonstrates the with specified siRNA. After an additional 2 d, protein and RNA were purified. (C) specificity of the IP. The four left-most lanes show threefold dilutions of WB was used to quantitate the extent of down-regulation. (D) RT-qPCR was 59 fi lysates from cells that expressed STAU2 -HA3.(B) RT-qPCR of FLUC mRNAs. used to quantitate the effects of tethering of the speci ed protein on FLUC- For samples before IP, the level of each FLUC mRNA was normalized to the MS2bs mRNA abundance. (E and F) Essentially as in C and D, except the effector R level of GAPDH mRNA, and the normalized level of FLUC mRNA from cells plasmids were siRNA resistant ( ). 55 that expressed STAU1 -HA3 was defined as 100. For samples after IP, the level of each immunoprecipitated FLUC mRNA was normalized to its nor- i malized level before IP. (C) After IP, samples were spiked with a small transfected with ( ) a pcFLUC-MS2bs reporter construct that amount of E. coli RNA, and RT-PCR measurements of the level of E. coli LacZ carries eight copies of the MS2 coat-protein binding sequence mRNA confirmed comparable RNA recovery efficiencies. (MS2bs) in its 3′ UTR (13), or, as a negative control, pcFLUC

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1213508110 Park et al. Downloaded by guest on September 29, 2021 (Fig. 5A), (ii) phCMV-MUP, and (iii) pMS2-HA-STAU155 (13), presence of poly(U) (Fig. 6G) or the RNA−DNA duplex (Fig. 62 59

pMS2-HA-STAU2 , or pMS2-HA-STAU2 , each of which S3E), none of the STAU1 or STAU2 isoforms or PABPC1 INAUGURAL ARTICLE encodes a tetherable protein. manifested appreciable ATPase activity itself or enhanced UPF1 The expression of each tetherable protein reduced the abun- ATPase activity. However, in the presence of ATP all STAU dance of FLUC-MS2bs reporter mRNA but not control FLUC isoforms, unlike PABPC1, activated UPF1 helicase activity in mRNA to ∼40–50% of normal (Fig. 5B). Using cells that were a concentration-dependent manner more or less to the same extent also transfected with Control or UPF1 siRNA (Fig. 5C), teth- (Fig. 6H and Fig. S3F; we consider the low level of helicase activity ering not only STAU155 (13, 14) but also STAU262 or STAU259 manifested by HIS-STAU155,STAU262,STAU259-HIS, or down-regulated FLUC-MS2bs reporter mRNA abundance to PABPC1 alone to be background activity). STAU1 or STAU2 did ∼40–60% of normal, and down-regulating UPF1 inhibited this not enhance UPF1 helicase activity in the absence of ATP (Fig. 6 down-regulation (Fig. 5 C and D). Thus, tethered STAU2, like I and J and Fig. S3 G and H) and also failed to activate UPF1 tethered STAU1, triggers SMD via its interaction with UPF1. (G495R,G497E) helicase activity (Fig. 6K). Using cells transfected with Control, STAU1, or STAU2 siRNA Results from our ATPase assays demonstrating that the STAU (Fig. 5E), tethering STAU155R, STAU262R, or STAU259R, each of paralog-mediated enhancement of UPF1 unwinding is not ac- which was produced from an siRNA-resistant vector, again re- companied by an increase in the number of ATP hydrolytic events duced FLUC-MS2bs reporter mRNA abundance to ∼20–40% of predicts that limiting ATP hydrolysis using NaVO3 to “trap” normal (Fig. 5F). STAU1 siRNA inhibited the ability of tethered UPF1 in a state that inhibits further hydrolysis would not inhibit STAU155R,STAU262R,orSTAU259R to reduce mRNA abundance, the effect of STAU155 on UPF1 unwinding. In fact, when present 55 as did STAU2 siRNA (Fig. 5F). Thus, neither STAU2 nor STAU1 in excess of ATP, NaVO3 does not inhibit the ability of STAU1 , can efficiently trigger SMD when the level of cellular STAU2, STAU262 and STAU259 to augment UPF1 helicase activity (Fig. 6 STAU1, or UPF1 is reduced, indicating that both paralogs con- L and M and Fig. S3 I–L). We propose that when UPF1 is bound tribute to SMD via their interaction with each other and UPF1. by ATP or a specific catalytic intermediate of ATP hydrolysis— one that is not recapitulated by any of the nucleotide analogs SMD Requires ATP-Dependent UPF1 Helicase Activity, Which Is tested here—STAU1, and possibly STAU2, promotes UPF1 Enhanced by STAU2 and STAU1 Without a Concomitant Enhancement helicase activity by inducing and/or stabilizing a helicase-active of UPF1 ATPase Activity. Binding of the UPF2 NMD factor to the conformation. UPF1 CH domain promotes UPF1 ATPase and helicase activi- ties in a way that is important for NMD (27, 28). Considering Evidence That STAU2 Induces a Conformational Change in RNA-Bound

that STAU1 and STAU2 bind to a region of UPF1 that contains UPF1. UPF2 augments UPF1 ATPase and helicase activities BIOCHEMISTRY the CH domain (15, 16), we tested the importance of UPF1 during NMD by binding to the UPF1 CH domain and, according ATPase and helicase activities to SMD by assaying UPF1 to RNase protection assays, decreasing the UPF1 footprint on (G495R,G497E) (29), which fails to bind ATP (30, 31). Human ssRNA (27). The decreased footprint was interpreted as a change UPF1 and UPF1(G495R,G497E) were produced in and purified in UPF1 conformation that loosened its grip on ssRNA, allowing from E. coli (Fig. S3A). ATPase activity assays were performed it to unwind RNA more effectively. in the presence of γ-[32P]ATP and, because the ATPase activity We performed comparable RNA footprint experiments to of UPF1 depends on RNA, poly(U) (30). The substrate for the determine whether STAU262 changes UPF1 conformation. We helicase assays consisted of a 44-nt ssRNA annealed to an 18-nt examined STAU262 because of its higher binding in vitro to UPF1 γ-[32P]-labeled ssDNA (28) (Fig. S3C), and assays were per- relative to STAU155 or STAU259 (Fig. 2B) and its greater solu- formed in the presence of excess unlabeled 18-nt ssDNA. In bility over STAU155 under footprinting conditions. STAU262 contrast to UPF1, UPF1(G495R,G497E) lacks both ATPase binding to UPF1 shifts the length of RNA protected by UPF1 A B C from 11 to 12 nt to 12 to 13 nt (Fig. 7A, compare lanes 2 and 3), (Fig. 6 ) and helicase (Fig. 6 and ) activities. − whereas in the absence of STAU262, ADPNP, ADP-AlF ,or Next, HEK293T cells were transiently transfected with − 4 a MYC-UPF1 or MYC-UPF1(G495R,G497E) expression vector ADP-VO3 binding to UPF1 increased the length of RNA pro- (29) and a subset of the pcFLUC SMD-reporter plasmids (Fig. tected to 12–14 nt (Fig. 7A, compare lanes 5–7), and ADP 1B). MYC-UPF1 proteins were each produced at a comparable binding further increased the length to 12–15 nt (Fig. 7A,lane9). level (Fig. 6D). Relative to MYC-UPF1, MYC-UPF1(G495R, We propose that STAU262 alters the conformation of UPF1 so G497E) inhibited SMD ∼two- to fourfold (Fig. 6E). Thus, UPF1 that it protects a length of RNA that is between the length pro- binding to ATP and the resulting ATPase and/or helicase ac- tected by ATP-free UPF1 and ATP (or an ATP-hydrolysis in- tivities are important for SMD because of roles in SMD-target termediate)-bound UPF1. Thus, STAU262 binding enables UPF1 remodeling and/or indirectly because of roles in UPF1 recycling. to approximate a conformation that is active in translocation To characterize the dependence of UPF1 helicase activity on and/or unwinding. In the absence of STAU262, the UPF1 foot- fi + ATP hydrolysis, helicase assays were performed in the presence print was not well de ned when ATP or ATP NaVO3 was of ATP; 5′-adenylyl β,γ-imidodiphosphate (ADPNP), which is a present (Fig. 7A, lanes 4 and 8) as expected because either − nonhydrolyzable analog of ATP; ADP-BeF , which mimics the UPF1 fails to bind RNA efficiently or the position of UPF1 on 3 − fi F M ground state of the ATP hydrolysis reaction (32); ADP-VO3 , RNA is not xed (Fig. 6 and ). Although not proven, we do which mimics the ADP+P transition state of ATP hydrolysis (33, not believe that the increased footprint observed when STAU262 i − 34); ATP together with NaVO , where VO replaces P after is added to UPF1 is due to STAU262 binding to ssRNA because 3 − 3 i 62 55 ATP hydrolysis to form ADP-VO3 at the active site and inhibit STAU2 , unlike STAU1 (2), does not appreciably bind our further hydrolysis (35–37); ADP; or no nucleotide. UPF1 heli- RNA−DNA duplex (Fig. S4 A and B), which contains 26 nt of case activity was evident in the presence of ATP and, to a lesser ssRNA. Moreover, STAU2 does not noticeably affect the ssRNA- extent, in the presence of ATP+NaVO but not in the absence of binding activity of UPF1 (Fig. S4B). The simplest interpretation 3 − − 62 ATP or the presence of ADPNP, ADP-BeF3 , ADP-VO3 ,or of our results is that the STAU2 -mediated increase in UPF1 ADP (Fig. 6F and Fig. S3D). Thus, limiting ATP hydrolysis using helicase activity is accompanied by an alteration in UPF1 con- B–D ATP+NaVO3 limits UPF1 helicase activity, and helicase activity is formation (Fig. 7 ). driven by the energy released from ATP cleavage and/or a result- ing cleavage-dependent change in UPF1 conformation that is not Discussion recapitulated by any of the tested nucleotide analogs. STAU2 Functions in SMD. Our results reveal that STAU2, like To determine the effect of STAU1 and STAU2 on UPF1 STAU1 (13), interacts directly with UPF1 both in vivo and in ATPase and helicase activities, the assays were repeated in the vitro, and these interactions are necessary for efficient SMD presence of increasing amounts of HIS-STAU155, STAU262, (Figs. 1, 2, and 5). Recently, Miki et al. (15) also demonstrated STAU259-HIS or, as a negative control, PABPC1. In the that STAU2 isoforms interact directly with UPF1 in vitro. In

Park et al. PNAS Early Edition | 5of8 Downloaded by guest on September 29, 2021 A B C D 120 UPF1 30 100 (G495R, UPF1 G497E) 25 80 - 20 Boiled 60 15 G497E) pCMV-MYC- 40 * 10

(% of UPF1) - UPF1(G495R, UPF1

Hydrolyzed ATP Hydrolyzed 20 5

Unwinding (%) Unwinding MYC-UPF1 0 * 0 (WB: -MYC) UPF1 UPF1 0 5 10 15 20 (nM) Calnexin (G495R, UPF1 (WB: G497E) UPF1(G495R,G497E) E -Calnexin) 600 F FLUC-GAP43 3’UTR mRNA 80 400 FLUC-c-JUN 60 SBS mRNA 40 200 FLUC-SERPINE1 20 FLUC mRNA / 0 Unwinding (%) Unwinding

RLUC mRNA (%) 3’UTR mRNA 0 ------UPF1 - UPF1 UPF1 (G495R, pCMV-MYC- - ATP + ADP ATP ADPNP ADP-- ADP-- ATP G497E) BeF VO G 3 3 NaVO3

300 200 Fig. 6. STAU155, STAU262, and STAU259 activate SMD 100 by augmenting UPF1 helicase activity but not UPF1

(% of UPF1) 0 ATPase activity. (A) Histogram of RNA-dependent

Hydrolyzed ATP Hydrolyzed ------++++++++ + --- +++ UPF1 ATPase assays using 10 nM of UPF1 or UPF1(G495R, STAU155 STAU262 STAU259 STAU155 STAU262 STAU259 PABPC1 PABPC1 G497E), where the activity of UPF1 was defined as 100. H μ γ 32 μ 100 Assays used 1 Ci of -[ P]ATP and 4 g of poly(U). (B) 75 ATP-dependent helicase assays using 0 (−), 2.5, 5, 10, or 50 20 nM (wedge) of UPF1 or UPF1(G495R,G497E) in the 25 presence of 0.25 nM of γ-[32P]-labeled RNA−DNA du- 0 Unwinding (%) Unwinding ------++++++++ + --- +++UPF1 plex. Mobilities of the RNA−DNA duplex and ssDNA are indicated by cartoons, where the asterisk (*) denotes STAU155 STAU262 STAU259 STAU155 STAU262 STAU259 PABPC1 PABPC1 γ-[32P]. (C) Plot of data in B.(D and E) HEK293T cells (4 × I 7 60 UPF1 10 per 150-mm dish) were transiently transfected with μ μ 40 K UPF1 (G495R,G497E) 1 g of each pcFLUC SMD-reporter plasmid; 0.5 gofthe μ − 20 pRLUC reference plasmid; and 1 g of pCMV-MYC( ), 55 62 59 55 62 59 pCMV-MYC-UPF1, or pCMV-MYC-UPF1(G495R,G497E).

Unwinding (%) Unwinding 0 + --+++- + --+++- --- ++ ++ UPF1 fi - - - 55 Two days later, protein and RNA were puri ed. (D)WB

STAU1 --STAU1 STAU2 STAU2 STAU1 STAU2 STAU2 using the specified antibody (α). The four left-most -ATP ATP ADP * J lanes show threefold dilutions of lysates from cells that 80 * expressed MYC-UPF1. (E) Histogram of RT-qPCR results. 60 The level of each FLUC mRNA was normalized to the 40 level of RLUC mRNA, and the normalized level of each 20 in pCMV-MYC-transfected cells was defined as 100. (F)

Unwinding (%) Unwinding 0 + ------+++++++ ------+++++++ ----++- - +++ +UPF1 62 59 62 59 62 59 62 59 62 59 62 59 As in B, using 0 (−), 10, 20, or 40 nM (wedge) of UPF1 in AU2 AU2 AU2 AU2 AU2 U2 AU2 AU2 AU2 AU2 U2 AU2 ST ST ST ST ST STA ST ST ST ST STA ST the absence of nucleotide (−ATP) or in the presence of − − -ATP ATPDA P0.5 mM of ATP, ADPNP, ADP-BeF3 , ADP-VO3 , or ADP, + L 100 or 0.5 mM of ATP together with ( ) 2.5 mM of NaVO3 80 (Fig. S3D). (G)AsinA, using 0 (−), 10, 20, or 40 nM of 60 + 40 UPF1 (wedge) or 10 nM UPF1 ( ) and/or 10, 20, or 40 20 nM of HIS-STAU155, STAU262, STAU259-HIS, or PABPC1 0 Unwinding (%) Unwinding + --+++- + --+++- --- ++ ++ UPF1 (wedge). (H) Helicase assays (Fig. S3F) using samples - - - 55 STAU1 assayed in G.(I and J)AsinB, using 0 (−)or10nMof + ATP ATP NaVO3 NaVO3 UPF1 (+) and/or 10, 20, or 40 nM (wedge) of (I) HIS- M 55 62 59 80 STAU1 ,(J) STAU2 or STAU2 -HIS in the absence of 60 nucleotide (−ATP) or in the presence of 2 mM of ATP or 40 ADP (Fig. S3 G and H). (K)AsinB, using 10 nM of UPF1 20 or UPF1(G495R,G497E) and 0 (−)or40nMofHIS-

Unwinding (%) Unwinding 0 + ------+++++++ ------+++++++ ----++- - +++ +UPF1 STAU155, STAU262, or STAU259-HIS. (L and M) Helicase 62 59 62 59 62 59 62 59 62 59 62 59 U2 U2 U2 U2 U2 U2 U2 U2 U2 U2 U2 U2 STA STA STA STA STA STA STA STA STA STA STA STA assays (Fig. S3 K and L)asinI and J, in the presence of + + 2 mM of ATP, 2 mM of ATP 10 mM of NaVO3,or10mM ATP ATP NaVO3 NaVO3 of NaVO3.

support of STAU2 function during SMD, tethering STAU2 STAU2−STAU2, STAU2−STAU1, and STAU1−STAU1 inter- downstream of the termination codon of a reporter mRNA, like actions are readily detected (Fig. 3) and may be required for op- tethering STAU1, reduces reporter mRNA abundance in timal UPF1 binding and/or activation. For example, our finding HEK293T cells in a mechanism that depends on UPF1, that STAU1 stabilizes imperfect intermolecular RNA duplexes STAU1, and STAU2 (Fig. 5). Additionally, the efficiency of that range in size from ∼85 to 300 bp (17) suggests that STAU SMD in tethering assays and assays of cellular targets is de- association contributes to duplex stabilization. Unlike Furic et al. termined by the abundance of both STAU1 and STAU2 (Fig. 5, (20), we find no evidence for STAU1- or STAU2-specificmRNA Fig. S5, and SI Discussion). Our finding that the two STAU targets (Fig. 4). Given that the paralogs interact, it is difficult to paralogs interact directly suggests that any functional differences conceive of such targets unless there were conditions whereby one between the two paralogs during SMD would be homogenized, paralog is significantly more abundant than the other. but less so for tissues in which one paralog is significantly more STAU1−STAU1 interactions have been shown using live abundant than the other. cells to involve dsRBD2 and dsRBD5 (22). Because STAU1

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1213508110 Park et al. Downloaded by guest on September 29, 2021 A dsRBD2 and parts of STAU1 dsRBD5 are conserved in all

− INAUGURAL ARTICLE UPF1 ++++++++ + STAU2 isoforms (Fig. 1), it is possible that STAU1 STAU2 and STAU262 ++ + STAU2−STAU2 interactions are also mediated by dsRBD2 and ATP + dsRBD5. Relative to STAU1, the binding of STAU2 to SBS- ADPNP + ∼ fi - containing mRNAs is two- to vefold higher (Fig. 4), which ADP-AlF4 + - + ADP-VO3 might be due to the additional dsRBD, dsRBD1, in STAU2 ATP+NaVO3 + compared with STAU1 (Fig. 1). However, because dsRBD3 and ADP + dsRBD4 seem to be the only functional dsRNA-binding domains 60-mer RNA ++++++++++++ + RNase A/T1 ++++++++++ (1, 2), contributions to RNA binding by dsRBD1 or by other 12345678910111213 nt differences between STAU2 and STAU1 might enhance STAU2 60 dsRBD3 and/or dsRBD4 function. For example, individual 50 40 dsRBDs that do not bind dsRNA in vitro can promote dsRNA binding of a “true” dsRBD when both reside in tandem as ex- 30 emplified by Xenopus laevis RNA-binding protein A (38). Deletion analyses indicate that STAU1 (16) and STAU2 (15) 20 interact with a region of UPF1 that includes the CH domain. nt This is the same region of UPF1 that interacts with UPF2 during 15 NMD. Relative to STAU1, the binding of STAU2 to UPF1 is 14 13 ∼10-fold higher (Fig. 2). Considering that residues spanning the 12 11 10 TBD to just upstream of dsRBD5 seem to be the minimal region of STAU1 that interacts with UPF1 (16), differences in the binding of STAU2 and STAU1 to UPF1 may be attributable to sequence variations in their TBDs, which share only 18% identity (Fig. 1). Provided the paralogs bind tightly enough to overcome

B UPF1 the activation threshold for UPF1 helicase activity, they may bind to overlapping or different regions of the CH domain with 1B CH STAU2 different efficiencies yet still activate UPF1 helicase activity to 1C A1A2 the same extent (Fig. 6). Alternatively, STAU2 may interact with

UPF1 using an entirely different region, such as dsRBD2 or BIOCHEMISTRY C dsRBD3 (15). ATP ATP ( ) hydrolysis ( ) RNA STAU2 Stimulates UPF1 Unwinding Activity but Not UPF1 ATP 11-12 nt-footprint 12-14 nt-footprint unwinding Hydrolytic Activity and Induces a Conformational Change in UPF1. D When added to UPF1, higher concentrations of STAU2 or STAU1 increase higher levels of UPF1 RNA−DNA duplex un- ATP ATP ( ) hydrolysis winding without stimulating UPF1 ATPase activity in a mecha- ( ) 12-13 nt-footprint STAU2- mediated nism that depends on the presence and/or hydrolysis of ATP enhanced (Fig. 6). There are two key differences between how STAU2 and RNA unwinding STAU1 activate UPF1 during SMD and how UPF2 activates Fig. 7. STAU262 expands the UPF1 footprint on ssRNA. (A)Thespecified UPF1 during NMD (27). First, UPF2 stimulates UPF1 ATPase complexes were formed using 5 pmol of in vitro-transcribed α-[32P]UTP- activity, whereas the STAU paralogs do not; they merely “grease labeled 60-nt RNA; 10 pmol of UPF1; 40 pmol of STAU262;andwhere the wheels” of the UPF1 helicase so that unwinding is more specified 2 mM of the indicated nucleotide and 10 mM of the specified 62 − extensive per ATP hydrolyzed. Second, at least for STAU2 , phosphate mimic. Notably, ADP-BeF3 was not tested because a pre- activation of UPF1 helicase activity is achieved by increasing − μ cipitate formed at 10 mM BeF3 . Samples were then incubated with 1 gof rather than decreasing the UPF1 footprint on RNA (Fig. 7). It RNase A and 1 U of RNase T1. The sizes of the protected RNA fragments has been reported that UPF2 in the presence of ADP-BeF3 were determined using denaturing polyacrylamide gel electrophoresis γ 32 decreases the size of the UPF1 footprint (27), UPF2 in the ab- followed by autoradiography. A -[ P]-labeled RNA ladder provided mo- sence of nucleotide analog loosens the grip of UPF1 on RNA lecular weight standards (black nt), and red notations denote protected fragments. Notably, sizes assigned to the protected fragments were con- (28), and UPF2 in the presence of ATP enhances UPF1 helicase firmed in gels, in which molecular weight standards were eletrophoresed activity (27). Although at pH 6.5 the footprint of UPF1 alone in lanes residing adjacent to each experimental lane. The nature of the (28) was found to be similar in size to the footprint of UPF1 in diffuse ∼6-nt band that was evident in the presence of all tested nucleo- the presence of ADPNP (27, 28), we are able to detect a differ- tides requires further investigation. The footprint in the presence of ADP- ence in the size of the two footprints at the more physiological − AlF4 was routinely faint. (B–D) Model for how STAU paralogs bind to and pH 7. We report here that UPF1 alone, which has a higher af- prolong a conformational state of UPF1 that promotes its ATP-dependent finity for ssRNA than when nucleotide bound (27, 31), has unwinding activity without promoting ATP hydrolysis. (B) Domains of a smaller footprint than when nucleotide bound. Therefore, the UPF1, where A1 and A2 represent the two RecA-like domains, 1B is an N- correlation of a smaller UPF1 footprint with activation of UPF1 terminal attachment of the A1 domain, 1C is an insertion in the A1 do- helicase activity, as observed in studies using UPF2 (27), may not main, and CH denotes the cysteine- and histidine-rich region (27, 31). (C) be universal, as evidenced from our studies using STAU2, which ATP-free UPF1 protects a smaller (11- to 12-nt) region of RNA than does indicate that a larger UPF1 footprint correlates with activation ATP-bound UPF1. The footprint of ATP-bound UPF1 consists of as many as of UPF1 helicase activity. Because nucleotide binding lowers the 14 nt and is maintained during the process of ATP hydrolysis and RNA association of UPF1 with RNA (27, 31), and a lowered associ- unwinding. Additional rounds of ATP hydrolysis lead to more extensive ation correlates with enhanced helicase activity (27), STAU262 duplex unwinding. (D) STAU2 association with UPF1 generates the larger binding to UPF1 may mimic nucleotide binding. UPF1 may re- UPF1 footprint that typifies UPF1 bound to ATP or a hydrolytic intermediate − semble the human coronavirus 229E helicase, which is capable of (e.g., ADPNP, ADP-VO3 , or ADP). Because STAU2 does not bind ssRNA, we infer that STAU2 alters the conformation of ssRNA-bound UPF1 (surrounding unwinding long stretches of duplexed RNA before dissociating yellow spikes) so UPF1 is better poised to receive ATP and/or unwind RNA (39), except that the processivity of UPF1 is augmented by its when ATP is already bound (surrounding green spikes). In this way, STAU2 binding to one or both STAU cofactors. enhances the intrinsic unwinding capability of UPF1 per ATP hydrolyzed. There are other examples of cofactor-mediated helicase acti- UPF1 helicase activity has been shown to scan and remodel mRNP during vation that seem to bypass coordinated ATPase stimulation. Ntr1 NMD (45, 46). of the Ntr1/Ntr2 complex enhances Prp43 RNA helicase activity

Park et al. PNAS Early Edition | 7of8 Downloaded by guest on September 29, 2021 in an ATP-dependent manner but without an apparent en- By analogy to the mechanism of NMD, after translation termi- hancement of ATPase activity (40, 41). As a variation of this nates sufficiently upstream of the SBS, STAU−UPF1 then theme, the E. coli Rep DNA helicase uses ATP hydrolysis not for reconfigures so that the helicase activity of UPF1 is activated unwinding but for translocation to a dsDNA structure and, by by STAU. analogy to how STAU cofactors activate the UPF1 helicase, the ØX174 gene A protein or constituents of the multi-Rep complex Materials and Methods then promote E. coli Rep helicase activity (42, 43). Taking cues Descriptions of plasmid constructions; cell transfection and lysis, and cell from these and other studies, we propose that STAU2 and protein and RNA purification are given in SI Materials and Methods. Sup- possibly STAU1 stabilize UPF1 in a conformation that is active porting Information also provides details for recombinant protein purifica- during unwinding and/or translocation (Fig. 7). tion and in vitro pull-down experiments. See SI Materials and Methods for in vitro ATPase and helicase assays; and RNase protection assays. Summary. We envision that STAU1 and/or STAU2 bind to an ACKNOWLEDGMENTS. We thank Susana de Lucas and Juan Ortín for anti- SBS either individually or, more likely, as homo- or hetero- STAU1, David Bear for the GST-PABPC1 plasmid, Yalan Tang for baculovirus- multimers, the size of which may depend on the length of the produced FLAG-UPF1, Galina Pavlencheva for E. coli-produced UPF1 and UPF1 SBS and the relative abundance of each paralog. STAU bound to (G495R,G497E), and Dmitri Ermolenko, Joseph Wedekind, Eckhard Jankowsky, an SBS recruits UPF1 either directly or through interactions with Hérve Le Hir, Fabien Bonneau, and L.E.M. laboratory members for advice. This work was funded by National Institutes of Health (NIH) Grant R01 GM074593 another STAU molecule, presumably after translation termi- (to L.E.M.). E.P. was supported by American Heart Association Founders Affil- nates if a comparison can be made to when UPF1 binds the stem- iate Postdoctoral Fellowship 11POST 7860051. M.L.G. was supported by NIH loop binding protein of cell cycle-regulated histone mRNAs (44). Grant F32 GM090479 and NIH National Cancer Institute Grant T32 CA09363.

1. Duchaîne TF, et al. (2002) Staufen2 isoforms localize to the somatodendritic domain 24. Hwang J, Sato H, Tang Y, Matsuda D, Maquat LE (2010) UPF1 association with the cap- of neurons and interact with different organelles. J Cell Sci 115(Pt 16):3285–3295. binding protein, CBP80, promotes nonsense-mediated mRNA decay at two distinct 2. Wickham L, Duchaîne T, Luo M, Nabi IR, DesGroseillers L (1999) Mammalian staufen is steps. Mol Cell 39(3):396–409. a double-stranded-RNA- and tubulin-binding protein which localizes to the rough 25. Hosoda N, Kim YK, Lejeune F, Maquat LE (2005) CBP80 promotes interaction of Upf1 . Mol Cell Biol 19(3):2220–2230. with Upf2 during nonsense-mediated mRNA decay in mammalian cells. Nat Struct 3. Monshausen M, Gehring NH, Kosik KS (2004) The mammalian RNA-binding protein Mol Biol 12(10):893–901. Staufen2 links nuclear and cytoplasmic RNA processing pathways in neurons. Neu- 26. Maher-Laporte M, et al. (2010) Molecular composition of staufen2-containing ribo- – romolecular Med 6(2-3):127 144. in embryonic rat brain. PLoS ONE 5(6):e11350. 4. Kanai Y, Dohmae N, Hirokawa N (2004) Kinesin transports RNA: Isolation and char- 27. Chakrabarti S, et al. (2011) Molecular mechanisms for the RNA-dependent ATPase – acterization of an RNA-transporting granule. Neuron 43(4):513 525. activity of Upf1 and its regulation by Upf2. Mol Cell 41(6):693–703. 5. Kiebler MA, et al. (1999) The mammalian staufen protein localizes to the somato- 28. Chamieh H, Ballut L, Bonneau F, Le Hir H (2008) NMD factors UPF2 and UPF3 bridge dendritic domain of cultured hippocampal neurons: Implications for its involvement UPF1 to the exon junction complex and stimulate its RNA helicase activity. Nat Struct in mRNA transport. J Neurosci 19(1):288–297. Mol Biol 15(1):85–93. 6. Thomas MG, et al. (2005) Staufen recruitment into stress granules does not affect 29. Isken O, Maquat LE (2008) The multiple lives of NMD factors: Balancing roles in gene early mRNA transport in oligodendrocytes. Mol Biol Cell 16(1):405–420. and genome regulation. Nat Rev Genet 9(9):699–712. 7. Vessey JP, et al. (2008) A loss of function allele for murine Staufen1 leads to im- 30. Bhattacharya A, et al. (2000) Characterization of the biochemical properties of the pairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis. Proc Natl Acad Sci USA 105(42):16374–16379. human Upf1 gene product that is involved in nonsense-mediated mRNA decay. RNA – 8. Tang SJ, Meulemans D, Vazquez L, Colaco N, Schuman E (2001) A role for a rat ho- 6(9):1226 1235. molog of staufen in the transport of RNA to neuronal dendrites. Neuron 32(3): 31. Cheng Z, Muhlrad D, Lim MK, Parker R, Song H (2007) Structural and functional in- – 463–475. sights into the human Upf1 helicase core. EMBO J 26(1):253 264. 9. Goetze B, et al. (2006) The brain-specific double-stranded RNA-binding protein 32. Gu M, Rice CM (2010) Three conformational snapshots of the hepatitis C virus NS3 Staufen2 is required for dendritic spine morphogenesis. J Cell Biol 172(2):221–231. helicase reveal a ratchet translocation mechanism. Proc Natl Acad Sci USA 107(2): 10. Lebeau G, et al. (2008) Staufen1 regulation of protein synthesis-dependent long-term 521–528. potentiation and synaptic function in hippocampal pyramidal cells. Mol Cell Biol 33. Lindquist RN, Lynn JL, Jr., Lienhard GE (1973) Possible transition-state analogs for 28(9):2896–2907. ribonuclease. The complexes of uridine with oxovanadium(IV) ion and vanadium(V) 11. Lebeau G, DesGroseillers L, Sossin W, Lacaille JC (2011) mRNA binding protein staufen ion. J Am Chem Soc 95(26):8762–8768. 1-dependent regulation of pyramidal cell spine morphology via NMDA receptor- 34. Westheimer FH (1987) Why nature chose phosphates. Science 235(4793):1173–1178. mediated synaptic plasticity. Mol Brain 4:22. 35. Goodno CC (1979) Inhibition of myosin ATPase by vanadate ion. Proc Natl Acad Sci 12. Lebeau G, et al. (2011) Staufen 2 regulates mGluR long-term depression and Map1b USA 76(6):2620–2624. mRNA distribution in hippocampal neurons. Learn Mem 18(5):314–326. 36. Goodno CC, Taylor EW (1982) Inhibition of actomyosin ATPase by vanadate. Proc Natl 13. Kim YK, Furic L, DesGroseillers L, Maquat LE (2005) Mammalian Staufen1 recruits Acad Sci USA 79(1):21–25. Upf1 to specific mRNA 3’UTRs so as to elicit mRNA decay. Cell 120(2):195–208. 37. Yamasaki K, Yamamoto T (1991) Existence of high- and low-affinity vanadate-binding 14. Kim YK, et al. (2007) Staufen1 regulates diverse classes of mammalian transcripts. sites on Ca(2+)-ATPase of the sarcoplasmic reticulum. J Biochem 110(6):915–921. EMBO J 26(11):2670–2681. 38. Krovat BC, Jantsch MF (1996) Comparative mutational analysis of the double- 15. Miki T, et al. (2011) Cell type-dependent gene regulation by Staufen2 in conjunction stranded RNA binding domains of Xenopus laevis RNA-binding protein A. J Biol Chem with Upf1. BMC Mol Biol 12:48. 271(45):28112–28119. 16. Gong C, Kim YK, Woeller CF, Tang Y, Maquat LE (2009) SMD and NMD are compet- 39. Ivanov KA, Ziebuhr J (2004) Human coronavirus 229E nonstructural protein 13: itive pathways that contribute to myogenesis: Effects on PAX3 and myogenin mRNAs. Characterization of duplex-unwinding, nucleoside triphosphatase, and RNA 5′-tri- Dev 23(1):54–66. phosphatase activities. J Virol 78(14):7833–7838. 17. Gong C, Maquat LE (2011) lncRNAs transactivate STAU1-mediated mRNA decay by 40. Tanaka N, Aronova A, Schwer B (2007) Ntr1 activates the Prp43 helicase to trigger duplexing with 3′ UTRs via Alu elements. Nature 470(7333):284–288. release of lariat-intron from the spliceosome. Genes Dev 21(18):2312–2325. 18. Cho H, et al. (2012) Staufen1-mediated mRNA decay functions in adipogenesis. Mol 41. Tsai RT, et al. (2005) Spliceosome disassembly catalyzed by Prp43 and its associated Cell 46(4):495–506. components Ntr1 and Ntr2. Genes Dev 19(24):2991–3003. 19. Gautrey H, McConnell J, Lako M, Hall J, Hesketh J (2008) Staufen1 is expressed in 42. Ha T, et al. (2002) Initiation and re-initiation of DNA unwinding by the Escherichia coli preimplantation mouse embryos and is required for embryonic stem cell differenti- Rep helicase. Nature 419(6907):638–641. ation. Biochim Biophys Acta 1783(10):1935–1942. 43. Yarranton GT, Gefter ML (1979) Enzyme-catalyzed DNA unwinding: studies on Es- 20. Furic L, Maher-Laporte M, DesGroseillers L (2008) A genome-wide approach identifies – distinct but overlapping subsets of cellular mRNAs associated with Staufen1- and cherichia coli rep protein. Proc Natl Acad Sci USA 76(4):1658 1662. Staufen2-containing ribonucleoprotein complexes. RNA 14(2):324–335. 44. Kaygun H, Marzluff WF (2005) Regulated degradation of replication-dependent – 21. Allison R, et al. (2004) Two distinct Staufen isoforms in Xenopus are vegetally local- histone mRNAs requires both ATR and Upf1. Nat Struct Mol Biol 12(9):794 800. ized during oogenesis. RNA 10(11):1751–1763. 45. Franks TM, Singh G, Lykke-Andersen J (2010) Upf1 ATPase-dependent mRNP disas- 22. Martel C, et al. (2010) Multimerization of Staufen1 in live cells. RNA 16(3):585–597. sembly is required for completion of nonsense- mediated mRNA decay. Cell 143(6): 23. Pal M, Ishigaki Y, Nagy E, Maquat LE (2001) Evidence that phosphorylation of human 938–950. Upfl protein varies with intracellular location and is mediated by a wortmannin- 46. Shigeoka T, Kato S, Kawaichi M, Ishida Y (2012) Evidence that the Upf1-related mo- sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway. RNA lecular motor scans the 3′-UTR to ensure mRNA integrity. Nucleic Acids Res 40(14): 7(1):5–15. 6887–6897.

8of8 | www.pnas.org/cgi/doi/10.1073/pnas.1213508110 Park et al. Downloaded by guest on September 29, 2021