Cdk5-Dependent Regulation of Rho Activity,Cytoskeletal Contraction,and Epithelial Cell Migration via Suppression of Src and p190RhoGAP

Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.Cell migration is essential for morphogenesis during embryonic development and for epithelial homeostasis and wound healing throughout life. As myosin II is involved in all aspects of cell migration, from cell polarization and adhesion to protrusion and tail retraction (34, 48), the signaling pathways regulating myosin-dependent cytoskeletal contraction are of particular interest. Myosin contraction is regulated by phosphorylation of myosin regulatory light chain (MRLC) at Thr18/Ser19. Although a number of kinases have been identified which phosphorylate these sites, the principal kinases in most cells are myosin light chain kinase (MLCK), a calcium/calmodulin-regulated enzyme, and Rho kinase (ROCK), a downstream effector of the Rho family GTPase RhoA. To provide the stringent control of cytoskeletal contraction needed for migration, RhoA is subject to both positive regulation by guanine nucleotide exchange factors (GEFs), such as GEF-H1 (4, 21), and negative regulation by GTPase-activating proteins (GAPs), such as the Src-regulated protein p190RhoGAP (1, 3, 10, 13). An additional level of regulation is provided by guanine nucleotide dissociation inhibitors, which bind to inactive RhoA and other Rho family GTPases, sequestering them in the cytosol (3). Two major downstream effectors of RhoA with regard to the cytoskeleton are the mammalian homologue of diaphanous, involved in actin polymerization (43), and ROCK, which phosphorylates MRLC and myosin phosphatase (20).Cdk5, a serine/threonine kinase, is an atypical member of the well-known family of cyclin-dependent kinases (Cdks). Unlike the other Cdks, it has no known function in cell cycle regulation and is activated by one of two noncyclin proteins, p35 or p39 (16, 41). Phosphorylation of Cdk5 at Y15 increases its activity severalfold (36, 49). Although Cdk5 is most abundant in neuronal cells, where it regulates migration, cytoskeletal dynamics, and membrane trafficking (37, 38, 45), a growing body of evidence indicates that Cdk5 has similar functions in nonneuronal cells (35). In particular, Cdk5 has been shown to strengthen cell-to-matrix adhesion and regulate migration in lens epithelial cells (28), corneal epithelial cells (11, 12, 40), keratinocytes (27), and CHO-K1 cells (15). The effects of Cdk5 on adhesion and migration have been linked, at least in part, to Cdk5-dependent phosphorylation of talin, which strengthens adhesion by slowing the rate of focal adhesion turnover (15). However, we have observed that Cdk5 not only binds to focal adhesions, where talin is located, but also to stress fibers (33). Moreover, in spreading cells, Cdk5 exerts its greatest effect on adhesion 1 to 2 h after plating (28), when stress fiber contraction is pronounced and Cdk5 activity is maximum (33). Therefore, we hypothesized that Cdk5 might regulate the MRLC phosphorylation necessary for stress fiber contraction and stability. To test this possibility, we examined the relationship of Cdk5 activity to MRLC phosphorylation and cytoskeletal contraction in spreading human lens epithelial cells.     

The Transition of Closely Opposed Lesions to Double-Strand Breaks during Long-Patch Base Excision Repair Is Prevented by the Coordinated Action of DNA Polymerase δ and Rad27/Fen1

DNA double-strand breaks can result from closely opposed breaks induced directly in complementary strands. Alternatively, double-strand breaks could be generated during repair of clustered damage, where the repair of closely opposed lesions has to be well coordinated. Using single and multiple mutants of Saccharomyces cerevisiae (budding yeast) that impede the interaction of DNA polymerase δ and the 5′-flap endonuclease Rad27/Fen1 with the PCNA sliding clamp, we show that the lack of coordination between these components during long-patch base excision repair of alkylation damage can result in many double-strand breaks within the chromosomes of nondividing haploid cells. This contrasts with the efficient repair of nonclustered methyl methanesulfonate-induced lesions, as measured by quantitative PCR and S1 nuclease cleavage of single-strand break sites. We conclude that closely opposed single-strand lesions are a unique threat to the genome and that repair of closely opposed strand damage requires greater spatial and temporal coordination between the participating proteins than does widely spaced damage in order to prevent the development of double-strand breaks.Endogenous metabolism or environmental factors such as oxidizing and alkylating agents can produce a wide variety of lesions in DNA. The genomes of mammalian cells experience from 10,000 to as many as 200,000 modifications per day (37, 44). Most lesions are repaired by a complex network of proteins that are part of an elaborate, multistep base excision repair (BER) system that generates single-strand break (SSB) intermediates. Importantly, defects in BER can lead to malignancies and can be associated with age-associated disease, especially neurodegeneration (60).BER is initiated by specific DNA N-glycosylases that remove damaged bases, yielding apurinic/apyrimidinic (AP) sites. Subsequent incision by AP endonucleases results in SSBs, and excision results in a single base gap as a repair intermediate (33, 53). SSBs are expected to be frequent in the genome due to the abundance of base damage as well as intermediates of repair, recombination, replication, and other DNA transactions (15, 16). Because they are generally repaired efficiently by BER and SSB repair enzymes (16, 57), SSBs per se may not be a major source of genome instability. However, if lesions are clustered, the formation of two closely spaced SSBs on opposing strands (or a single SSB and a modified nucleotide or AP site) might pose a special risk in terms of the potential to generate mutations or the possibility of conversion to double-strand breaks (DSBs), which are potent genotoxic lesions. Clustered lesions can arise within cells by chance association of random DNA lesions in a small region or the induction of multiple events in a narrow region, as found for ionizing radiation and various chemicals, such as those used in cancer treatments (47, 58, 59). While efficient BER is important for genome integrity, the repair must be well coordinated to avoid the generation of closely opposed SSB intermediates at closely spaced lesions that could result in the secondary generation of DSBs, especially since cells have limited DSB repair capacity (<50 DSBs/cell in the case of Saccharomyces cerevisiae) (48). While the impact of clustered lesions on repair of DNA has been examined in vitro by use of purified enzymes or cell extracts (13, 14, 27, 39, 56), there has been little opportunity to address specifically the repair of clustered lesions, except for those arising from UV damage (49).Whether formed directly from sugar damage or as BER intermediates, SSBs formed during the repair of base damage often possess 5′-deoxyribose phosphate (5′-dRP) ends that are not suitable for rejoining by DNA ligases (9, 15). In humans, removal and repair of 5′-dRP are accomplished by different combinations of proteins (3, 15) that result in short-patch repair, involving replacement of a single nucleotide (nt), or long-patch repair, involving 2 to 10 nt. The budding yeast Saccharomyces cerevisiae lacks a DNA polymerase β that provides AP lyase activity required for short-patch repair in mammalian cells. Instead, removal and repair of a 5′-dRP rely on the long-patch pathway, involving the successive actions of DNA polymerase δ (Pol δ) for strand displacement, the Rad27/Fen1 endonuclease to remove 5′ flaps, and DNA ligase (Cdc9) to rejoin the resulting nicks (9). The sliding clamp protein PCNA, which interacts with all three players, has been proposed to play a central role in coordinating these processes (18, 19, 34). The coupling between the strand displacement reaction by Pol δ and the flap cutting reaction by Fen1 is highly efficient, with over 90% of the products released by Fen1 being mononucleotides (17).Although the coordination of Pol δ, PCNA, and Rad27/Fen1 provides efficient processing of individual lesions in DNA, closely opposed SSBs that arise during repair of base damage could manifest as DSBs, either directly or as a result of SSB processing. A DNA damaging agent that has been used frequently to characterize long- and short-patch BER is methyl methanesulfonate (MMS). Recently, we described the detection of closely opposed MMS-induced lesions in yeast (42). Since the closely opposed lesions might represent a special challenge to BER, we considered the possibility that they might specifically impact long-patch repair through Pol δ and/or coordination of events with Rad27/Fen1. Pol δ of S. cerevisiae is a heterotrimeric enzyme consisting of Pol3, Pol31, and Pol32 (23). The nonessential Pol32 subunit is involved in translesion DNA synthesis (TLS) (24, 30) and also break-induced replication (41). However, its role in other types of DNA repair remains unclear. Using our in vivo assay for specifically detecting closely spaced methylated DNA lesions (42) and SSBs, we examined the role of Pol32 as well as the cooperation between Pol δ, Rad27/Fen1, and PCNA in the repair of clustered DNA lesions induced by MMS in G₁ stationary-phase haploid yeast. We found that Pol32 plays an important role in ensuring that clustered lesions are efficiently repaired and do not transition to DSBs.     

Finger Domain Mutation Affects Enzyme Activity,DNA Replication Efficiency,and Fidelity of an Exonuclease-Deficient DNA Polymerase of Herpes Simplex Virus Type 1

The catalytic subunit of herpes simplex virus DNA polymerase (Pol), a member of the B family polymerases, possesses both polymerase and exonuclease activities. We previously demonstrated that a recombinant virus (YD12) containing a double mutation within conserved exonuclease motif III of the Pol was highly mutagenic and rapidly evolved to contain an additional leucine-to-phenylalanine mutation at residue 774 (L774F), which is located within the finger subdomain of the polymerase domain. We further demonstrated that the recombinant L774F virus replicated DNA with increased fidelity and that the L774F mutant Pol exhibited altered enzyme kinetics and impaired polymerase activity to extension from mismatched primer termini. In this study, we demonstrated that addition of the L774F mutation to the YD12 Pol did not restore the exonuclease deficiency. However, the polymerase activity of the YD12 Pol to extension from mismatched primer termini and on the nucleotide incorporation pattern was altered upon addition of the L774F mutation. The L774F mutation-containing YD12 Pol also supported the growth of viral progeny and replicated DNA more efficiently and more accurately than did the YD12 Pol. Together, these studies demonstrate that a herpes simplex virus Pol mutant with a highly mutagenic ability can rapidly acquire additional mutations, which may be selected for their survival and outgrowth. Furthermore, the studies demonstrate that the polymerase activity of HSV-1 Pol on primer extension is influenced by sequence context and that herpes simplex virus type 1 Pol may dissociate more frequently at G·C sites during the polymerization reaction. The implications of the findings are discussed.Herpes simplex virus (HSV) DNA polymerase consists of the catalytic subunit of the polymerase (Pol) and the processivity factor UL42. The Pol subunit contains three well-defined activities: polymerization (replication), exonuclease proofreading (editing), and UL42 binding (5, 6, 28). The UL42 binding activity is mediated by amino acid residues located at the C terminus (5, 6). Although the UL42 binding residues are unique to certain alphaherpesvirus DNA polymerases, the sequences comprising the polymerase and exonuclease domains are conserved among the B family (or the α-like) polymerases (2-4). The exonuclease domain of the HSV type 1 (HSV-1) Pol contains conserved exonuclease I (Exo I), II, and III motifs, whereas the polymerase domain contains seven conserved regions (I to VII); conserved region IV overlaps with the Exo II motif. The Exo III motif is located within the δ region C, which is highly conserved among the B family polymerases (Fig. ​(Fig.1A).1A). These conserved regions are located within the palm, the thumb, and the finger subdomains, which comprise the structural components of the polymerase domain. The crystal structure of the HSV-1 Pol subunit revealed three grooves that form the putative polymerase, exonuclease, and DNA binding sites. The putative exonuclease site is defined as a groove formed between the exonuclease domain and the tip of the thumb subdomain. The palm and thumb subdomains form a groove proposed to be the putative duplex DNA binding site for both the editing and the polymerization complexes (23). Thus, the polymerase and exonuclease domains of HSV-1 are structurally and functionally interconnected (1, 7, 16, 21, 23, 27, 28), although they are organized into two different domains.Open in a separate windowFIG. 1.(A) Schematic diagram of the conserved regions and motifs within HSV-1 Pol. The relative locations of the conserved regions of HSV-1 Pol are shown at the top; regions I to VII and δ region C are represented by open boxes. The conserved exonuclease motifs I, II, and III are indicated with closed boxes. The functional and structural domains (determined by crystal structure analysis [23]) of the HSV-1 Pol are shown below. The N-terminal domain (N domain) is composed of two regions separated by the 3′-to-5′ exonuclease domain (23). (B) Schematic diagram of wild-type and mutant YDL Pol. The BamHI fragment of the wild-type pol from the plasmid pHC629 is shown at the top. The relative location of conserved region VI and the Exo III motif are shown below, with corresponding wild-type and mutant (YDL) amino acid sequences. B, BamHI; M, MstI; N, NotI.The high fidelity of DNA replication is achieved by three different mechanisms: nucleotide discrimination during the polymerization reaction, editing immediately after the polymerization reaction, and postreplication repair. HSV-1 mutant Pol containing mutations within the conserved regions of the polymerase domain can result in altered enzyme kinetics and DNA replication fidelity (8, 9, 11, 12, 18, 26). Similarly, mutation of conserved Exo domain residues can lead to the loss of exonuclease activity and to altered nucleotide selection and incorporation kinetics as well as the mutator phenotype (1, 10, 13, 14, 21, 25). Our previous studies demonstrated that a mutant Pol (YD12) containing a tyrosine-to-histidine substitution at residue 577 and an aspartic acid-to-alanine substitution at residue 581 (Y577H/D581A) is exonuclease deficient (exo⁻) and that recombinant virus expressing the mutant Pol exhibits a mutator phenotype in vivo (14). However, this recombinant virus rapidly evolved to contain an additional leucine-to-phenylalanine substitution at residue 774 (L774F), which is located within conserved region VI of the polymerase domain (18). Interestingly, a recombinant virus containing the L774F Pol mutation exhibits increased fidelity of DNA replication (18). Our recent study also demonstrated that the mutant L774F Pol exhibits altered enzyme kinetics (26). These results led to the hypothesis that the emerged L774F mutation in the context of the YD12 Pol mutant may also affect enzyme activity, DNA replication, and fidelity.     

Functional and Spatial Regulation of Mitotic Centromere- Associated Kinesin by Cyclin-Dependent Kinase 1

Mitotic centromere-associated kinesin (MCAK) plays an essential role in spindle formation and in correction of improper microtubule-kinetochore attachments. The localization and activity of MCAK at the centromere/kinetochore are controlled by Aurora B kinase. However, MCAK is also abundant in the cytosol and at centrosomes during mitosis, and its regulatory mechanism at these sites is unknown. We show here that cyclin-dependent kinase 1 (Cdk1) phosphorylates T537 in the core domain of MCAK and attenuates its microtubule-destabilizing activity in vitro and in vivo. Phosphorylation of MCAK by Cdk1 promotes the release of MCAK from centrosomes and is required for proper spindle formation. Interfering with the regulation of MCAK by Cdk1 causes dramatic defects in spindle formation and in chromosome positioning. This is the first study demonstrating that Cdk1 regulates the localization and activity of MCAK in mitosis by directly phosphorylating the catalytic core domain of MCAK.Chromosomes are properly attached to the mitotic spindles, and chromosome movement is tightly linked to the structure and dynamics of spindle microtubules during mitosis. Important regulators of microtubule dynamics are the kinesin-13 proteins (37). This kinesin superfamily is defined by the localization of the conserved kinesin core motor domain in the middle of the polypeptide (19). Kinesin-13 proteins induce microtubule depolymerization by disassembling tubulin subunits from the polymer end (6). Among them, mitotic centromere-associated kinesin (MCAK) is the best-characterized member of the family. It depolymerizes microtubules in vitro and in vivo, regulates microtubule dynamics, and has been implicated in correcting misaligned chromosomes (12, 14, 16, 24). In agreement with these observations, both overexpression and inhibition of MCAK result in a disruption of microtubule dynamics, leading further to improper spindle assembly and errors in chromosome alignment and segregation (7, 11, 15, 22, 33). The importance of MCAK in ensuring the faithful segregation of chromosomes is consistent with the observation that MCAK is highly expressed in several types of cancer and thus is likely to be involved in causing aneuploidy (25, 32).While MCAK is found both in the cytoplasm and at the centromeres throughout the cell cycle, it is highly enriched on centrosomes, the centromeres/kinetochores, and the spindle midzone during mitosis (18, 21, 36, 38). In accordance with its localizations, MCAK affects many aspects throughout mitosis, from spindle assembly and maintenance (3, 10, 36) to chromosome positioning and segregation (14, 21, 35). Thus, the precise control of the localization and activity of MCAK is crucial for maintaining genetic integrity during mitosis. Regulation of MCAK on the centromeres/kinetochores by Aurora B kinase in mitosis has been intensively investigated (1, 28, 29, 43). The data reveal that MCAK is phosphorylated on several serine/threonine residues by Aurora B, which inhibits the microtubule-destabilizing activity of MCAK and regulates its localization on chromosome arms/centromeres/kinetochores during mitosis (1, 18, 28). Moreover, in concert with Aurora B, ICIS (inner centromere KinI stimulator), a protein targeting the inner centromeres in an MCAK-dependent manner, may regulate MCAK at the inner centromeres and prevent kinetochore-microtubule attachment errors in mitosis by stimulating the activity of MCAK (27). Interestingly, hSgo2, a recently discovered inner centromere protein essential for centromere cohesion, has been reported to be important in localizing MCAK to the centromere and in spatially regulating its mitotic activity (13). These data highlight that the activity and localization of MCAK on the centromeres/kinetochores during mitosis are tightly controlled by Aurora B and its cofactors. Remarkably, MCAK concentrates at spindle poles from prophase to telophase during mitosis (18); however, only a few studies have been done to deal with that issue. Aurora A-depleted prometaphase cells delocalize MCAK from spindle poles but accumulate the microtubule-stabilizing protein ch-TOG at poles (5), implying that Aurora A might influence the centrosomal localization of MCAK in mitosis. Aurora A is also found to be important for focusing microtubules at aster centers and for facilitating the transition from asters to bipolar spindles in Xenopus egg extracts (42). In addition, it has been revealed that Ca²⁺/calmodulin-dependent protein kinase II gamma (CaMKII gamma) suppresses MCAK''s activity, which is essential for bipolar spindle formation in mitosis (11). More work is required to gain insight into the regulatory mechanisms of MCAK at spindle poles during mitosis.Deregulated cyclin-dependent kinases (Cdks) are very often linked to genomic and chromosomal instability (20). Cyclin B1, the regulatory subunit of Cdk1, is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment (2, 4). We observed that knockdown of cyclin B1 induces defects in chromosome alignment and mitotic spindle formation (N.-N. Kreis, M. Sanhaji, A. Krämer, K. Sommor, F. Rödel, K. Strebhardt, and J. Yuan, submitted for publication). Yet, how Cdk1/cyclin B1 carries out these functions is not very well understood. In this context, it is extremely interesting to investigate the relationship between the essential mitotic kinase Cdk1 and the microtubule depolymerase MCAK in human cells.     

Interaction of decay-accelerating factor with echovirus 7

Echovirus 7 (EV7) belongs to the Enterovirus genus within the family Picornaviridae. Many picornaviruses use IgG-like receptors that bind in the viral canyon and are required to initiate viral uncoating during infection. However, in addition, some of the enteroviruses use an alternative or additional receptor that binds outside the canyon. Decay-accelerating factor (DAF) has been identified as a cellular receptor for EV7. The crystal structure of EV7 has been determined to 3.1-Å resolution and used to interpret the 7.2-Å-resolution cryo-electron microscopy reconstruction of EV7 complexed with DAF. Each DAF binding site on EV7 is near a 2-fold icosahedral symmetry axis, which differs from the binding site of DAF on the surface of coxsackievirus B3, indicating that there are independent evolutionary processes by which DAF was selected as a picornavirus accessory receptor. This suggests that there is an advantage for these viruses to recognize DAF during the initial process of infection.Echoviruses (EVs) belong to the family Picornaviridae, which contains some of the most common viral pathogens of vertebrates (43, 50, 51, 55, 58, 63). Picornaviruses are small, icosahedral, nonenveloped animal viruses. Their capsids have 60 copies each of four viral proteins, VP1, VP2, VP3, and VP4, that form an ∼300-Å-diameter icosahedral shell filled with a positive-sense, single-stranded RNA genome. A distinctive feature of the capsid surface is a depression around the 5-fold axes of symmetry, called the “canyon” (47). The results of both genetic and structural studies have shown that the canyon is the site of receptor binding for many of these viruses (4, 11, 23, 25, 36, 47, 68), including echoviruses, which utilize β-integrins (6, 33, 66). Receptor molecules that bind in the canyon have been found to belong to the immunoglobulin superfamily (49). When these receptor molecules bind within the canyon, they dislodge a “pocket factor” within a pocket immediately below the surface of the canyon. The shape and environment of the pocket factor suggest that it might be a lipid (13, 32, 45, 54). When a receptor binds within the canyon, it depresses the floor of the canyon, corresponding to the roof of the pocket. Similarly, when a lipid or antiviral compound binds to the pocket, it expands the roof of the pocket, corresponding to the floor of the canyon (39, 45). Thus, receptors that bind to the canyon and the pocket factor compete with each other for binding to the virus. An absence of the hydrophobic pocket factor destabilizes the virus and initiates transition to altered “A” particles, a likely prelude to uncoating of the virion, possibly during passage through an endosomal vesicle (45).Not all receptors of picornaviruses bind in the canyon. A minor group of human rhinoviruses (HRV) bind to the low-density-lipoprotein receptor family (17, 34, 61, 62), and some other picornaviruses, including certain coxsackie- and echoviruses, utilize decay-accelerating factor (DAF; also called CD55) as a cellular receptor (9, 28, 40, 52).DAF is a member of a family of proteins that regulate complement activation by binding to and accelerating the decay of both classical and alternative pathway C3 and C5 convertases (7, 18, 26), the central amplification enzymes of the complement cascade. DAF is expressed on virtually all cell surfaces, protecting self cells from the immune system by rapidly dissociating any convertases that assemble, thereby halting the progression of a complement attack directed at the cell. Recent work (15, 27, 29, 56) has shown that DAF also participates in T-cell antiviral immunity (56) and protects against T-cell autoimmunity (29) by regulating complement that is produced locally by immune cells. The functional region of DAF consists of four short consensus repeats (SCR1, -2, -3, and -4). The structures have been determined for the SCR2-SCR3 fragment, the SCR3-SCR4 fragment, and the full four-domain region (30, 60, 65). Each of the SCR domains contains about 60 residues and is folded into a β structure stabilized by disulfide bridges. The four SCR domains form a relatively rigid extended rod with dimensions of 160 by 50 by 30 Å (30). The four domains rise about 180 Å above the plasma membrane, on a serine- and threonine-rich stalk of 94 amino acids, 11 of which are O-glycosylated, and is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor.Structural and genetic studies have shown that closely related picornaviruses have adapted to bind to DAF at different sites on the receptor surface (9, 31, 38, 42, 52, 64). Although DAF binding is likely to facilitate viral adsorption, the availability of DAF receptor molecules on the host is normally not sufficient for echovirus 7 (EV7) to enter cells. Presumably, viral adaptation to bind DAF offers some advantage to the virus, such as increasing the efficiency of infection.In an earlier publication (14), a 16-Å-resolution cryo-electron microscopy (cryo-EM) density map of the EV7-DAF complex was interpreted with the homologous structures of coxsackievirus B3 (CVB3) for EV7 (74% sequence identity) and virus complement protein for DAF (25% sequence identity). Because of the limited resolution of the earlier cryo-EM reconstruction, it was concluded that DAF bound to EV7 by laying across the icosahedral 2-fold axes. This implied that there were two alternative DAF binding modes occupying the same site, but with DAF oriented in opposite directions, and that only one of these alternative sites could be occupied at a time. Here we describe an improved, 7.2-Å-resolution cryo-EM reconstruction of DAF bound to EV7 and 3.1-Å-resolution X-ray crystal structures of EV7. Together with previously determined structures of DAF (30), we now show that 2-fold axis-related DAF molecules bind close to the icosahedral 2-fold axes on the viral surface but (in contradiction to the earlier results and consistent with predictions made by Pettigrew et al. [38]) do not cross these axes. This is consistent with the results of DAF binding to EV12, which binds DAF similarly to the manner reported here and also predicted for EV7 (38). Thus, the binding modes of DAF to EV12 and EV7 are now shown to be similar, but not the same, and are completely different from the binding mode of DAF to CVB3.     

A Targeted Bypass Screen Identifies Ynl187p,Prp42p,Snu71p,and Cbp80p for Stable U1 snRNP/Pre-mRNA Interaction

To understand how DEXD/H-box proteins recognize and interact with their cellular substrates, we have been studying Prp28p, a DEXD/H-box splicing factor required for switching the U1 snRNP with the U6 snRNP at the precursor mRNA (pre-mRNA) 5′ splice site. We previously demonstrated that the requirement for Prp28p can be eliminated by mutations that alter either the U1 snRNA or the U1C protein, suggesting that both are targets of Prp28p. Inspired by this finding, we designed a bypass genetic screen to specifically search for additional, novel targets of Prp28p. The screen identified Prp42p, Snu71p, and Cbp80p, all known components of commitment complexes, as well as Ynl187p, a protein of uncertain function. To examine the role of Ynl187p in splicing, we carried out extensive genetic and biochemical analysis, including chromatin immunoprecipitation. Our data suggest that Ynl187p acts in concert with U1C and Cbp80p to help stabilize the U1 snRNP-5′ splice site interaction. These findings are discussed in the context of DEXD/H-box proteins and their role in vivo as well as the potential need for more integral U1-snRNP proteins in governing the fungal 5′ splice site RNA-RNA interaction compared to the number of U1 snRNP proteins needed by metazoans.Nuclear precursor mRNA (pre-mRNA) splicing takes place in the spliceosome, a large dynamic complex consisting of over 100 proteins and five small nuclear RNAs (snRNAs) (32, 70). During spliceosome assembly, the U1 small nuclear ribonucleoprotein particle (snRNP) first contacts the pre-mRNA 5′ splice site (5′ss), followed by binding of the U2 snRNP to the branch site and the joining of the U5-U4/U6 tri-snRNP (32, 64, 70). The step in which U1 snRNP binds to the 5′ss is arguably one of the most critical, because it probably commits pre-mRNA to the splicing pathway (38, 48, 49, 60, 74). In the budding yeast Saccharomyces cerevisiae in vitro system, two U1-snRNP-containing commitment complexes (CCs), CC1 and CC2, can be detected by native gel electrophoresis prior to the U2 snRNP''s joining to form the prespliceosome (38, 60). CC1, whose formation is dependent on a functional 5′ss, appears to be a kinetic precursor to CC2, whose formation requires both a functional 5′ss and branch site and the participation of the branch-site-binding protein (BBP) and Mud2p, which are likely equivalent to SF1 and U2AF65, respectively, in the mammalian system (1-3, 75).Accumulating evidence suggests that formation of the canonical 5- to 7-bp RNA duplex between U1 snRNA and the 5′ss region is not sufficient to cause a stable CC to form in the yeast system (59, 62, 78); protein-RNA contacts are also important. For example, Zhang and Rosbash (77) identified eight proteins, all present in CCs, that make physical contact with the pre-mRNA at or near the 5′ss. Four of these proteins, U1C, U1-70K, Snu56p, and Nam8p, are integral parts of the U1 snRNP (20), and another three, SmB, SmD1, and SmD3, belong to the seven-member ring that binds the conserved Sm site present on U1, U2, U4, and U5 snRNAs (33, 71). The remaining protein, Cbp80p, is a subunit of the nuclear cap-binding complex (CBC), which also contains a second subunit, Cbp20p (28, 39). Interestingly, despite being a non-snRNP factor, Cbp80p is known to collaborate with U1 snRNP to help form or stabilize CC1 (8, 40). Furthermore, the contact between the C-terminal tails of SmB, SmD1, and SmD3 and the pre-mRNA may contribute to stabilizing the U1 snRNP/pre-mRNA interaction (76). Finally, Du and Rosbash (11) more recently showed that U1C is capable of selecting splice-site-like sequences in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast splice-site consensus sequence.Once fully assembled, the spliceosome must progress through a number of major structural and conformational changes to form the catalytic center; these include a series of highly orchestrated RNA-RNA rearrangements (53, 64, 70). Some of these are mutually exclusive; i.e., the formation of one RNA duplex requires the disruption of another. For example, the base-pairing interaction between the U1 snRNA and the 5′ss is replaced by a U6 snRNA/5′ss pairing. This exchange appears to be coupled to U4/U6 RNA unwinding (53, 64, 70). It is now known that splicing factors belonging to the ATPase II superfamily (18), which are also termed the DEXD/H-box proteins (5, 43), promote spliceosomal RNA rearrangements (64). However, the precise roles of most DEXD/H-box proteins remain unclear.It has been nearly 2 decades since DEXD/H-box proteins were first proposed to be RNA helicases (44). Over the years, a wealth of data revealed that DEXD/H-box proteins are essential in most, if not all, RNA-related pathways, e.g., splicing, mRNA export, and ribosomal biogenesis (5, 43, 64). Their modes of action in vivo remain a mystery, however. For example, Lorsch and Herschlag (45, 46) proposed that DEXD/H-box proteins may perform functions which are distinct from RNA unwinding and include mediating large-scale RNA structural rearrangements, disrupting protein-RNA or protein-protein interactions, and functioning as fidelity sensors in RNA-RNA interactions and rearrangements. Indeed, recent data confirm that DEXD/H-box proteins can catalyze protein displacement in a manner independent of RNA duplex unwinding (30). Therefore, the essential functions of DExH/D proteins can be exerted on a wide range of RNP substrates. This “RNPase” (or ATPase for RNP remodeling) hypothesis appears especially attractive in light of the fact that RNA duplexes in vivo are rarely more than ∼10 contiguous base pairs in length and that they often require protein binding for stabilization (21, 63). To fully understand how DEXD/H-box proteins function in the cell, it is critical to identify their physiological substrates.Inspired by our previous finding that the requirement for Prp28p, an essential DEXD/H-box splicing factor, can be bypassed by mutations that alter the YHC1 gene, which encodes U1C protein (7), we sought to exploit the bypass concept to deepen our understanding of the role of Prp28p in splicing. The underlying hypothesis is that bypass mutations define gene products that Prp28p may counteract. Here we describe the outcome of this approach and provide a detailed analysis of Ynl187p, a novel protein that probably contributes to stabilizing the U1 snRNP-5′ss interaction.