However, this wide glycan binding spectrum in a unique binding mode of the GII.13/21 huNoVs appears not to increase their prevalence, probably due to the existence of decoy glycan receptors in human gastrointestinal tract limiting their infection. Our findings shed light on the host interaction and epidemiology of huNoVs, which would impact the strategy of huNoV control and prevention.Evidence from both phenotypic binding assay and structural study support the observed interactions of human noroviruses (huNoVs) with histo-blood group antigens (HBGAs) as receptors or attachment factors, affecting their host susceptibility. GII.13 and GII.21 genotypes form a unique genetic lineage that differs from the mainstream GII huNoVs in their unconventional glycan binding site. Unlike the previous findings that GII.13/21 genotypes recognize only Le a antigen. This previous finding raised the question of whether the new GII.13/21 GBS really has such a narrow glycan binding spectrum. In this study, we provide solid phenotypic and structural evidence indicating that this new GBS recognizes a group of glycans with a common terminal β-galactose . First, we found that P domain proteins of GII.13/21 huNoVs circulating at different times bound three glycans sharing a common terminal β-Gal, including Lec, lactose, and mucin core 2. Second, we solved the crystal structures of the GII.13 P dimers in complex with Lec and mucin core 2, which showed that β-Gal is the major binding saccharide. Third, nonfat milk and lactose blocked the GII.13/21 P domain-glycan binding, which may explain the low prevalence of GII.13/21 viruses. Our data provide new insight into the host interactions and epidemiology of huNoVs, which would help in the control and prevention of NoV-associated diseases.Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as host susceptibility factors. GII.13 and GII.21 huNoVs form a unique genetic lineage that emerged from mainstream GII NoVs via development of a new, nonconventional glycan binding site (GBS) that binds LeIMPORTANCE Evidence from both phenotypic binding assay and structural study support the observed interactions of human noroviruses (huNoVs) with histo-blood group antigens (HBGAs) as receptors or attachment factors, affecting their host susceptibility. GII.13 and GII.21 genotypes form a unique genetic lineage that differs from the mainstream GII huNoVs in their unconventional glycan binding site. Unlike the previous findings that GII.13/21 genotypes recognize only Lea antigen, we found in this study that they can interact with a group of glycans with a common terminal β-Gal, including Lec, lactose, and mucin core 2. However, this wide glycan binding spectrum in a unique binding mode of the GII.13/21 huNoVs appears not to increase their prevalence, probably due to the existence of decoy glycan receptors in human gastrointestinal tract limiting their infection. Our findings shed light on the host interaction and epidemiology of huNoVs, which would impact the strategy of huNoV control and prevention. Norovirus genus of the Caliciviridae family, are the leading cause of acute gastroenteritis (AGE) and are associated with almost 20% of all AGE cases worldwide (Human noroviruses (huNoVs), members of the orldwide . NoVs arNoVs are classified into seven genogroups (GI to GVII), among which GI, GII, and GIV cause human infections . HuNoVs 3–In addition to HBGAs, huNoVs can also recognize other glycans or small chemicals. For example, some huNoVs (GI.3 VA115 and GII.4 VA387) reportedly bind gangliosides and sialic acid-containing glycoconjugates . On the 14–a antigen of HBGAs , points away from the surface of the P dimer and as a result, only a hydrophobic interaction was formed with E396.To further explore the structural basis of the observed GII.13 huNoV-glycan interaction, we solved the crystal structure of the GII.13 SC1065/2016 P domain in complex with Lec disaccharide to a 1.6-Å resolution in the P12ce group . Lec disce group , and twoce group . Eight rce group . Specifice group  and D. IThe crystal structure of the GII.13 08N2045/2008 P domain in complex with mucin core 2 trisaccharide was also determined at a 1.7-Å resolution . All thra antigen , but it was a tyrosine (Y) in GII.21 OIF, while G361 in the GII.13 SC1065 changed to an E358 in GII.21 OIF. Because the GII.21 OIF P domain was reported to bind Le antigen  but the  antigen , aiming  antigen . Also, t antigen .a in GII.21 OIF  and GIIConsistent with the binding to lactose, nonfat milk blocked the glycan binding function of the GII.13 and GII.21 P proteins  and B buSimilarly, a lactose blocking assay showed that free Lac oligosaccharides blocked binding of the GII.13 and GII.21 P domain proteins to Lec, mucin core 2-PAA conjugate  and B, aa antigen, which is one of the many HBGAs . The saliva samples from 225 children were collected previously and stored in our laboratory. The children were between 3 and 8 years old, and the ratio of boys to girls was 1.5:1; some of these children suffered from NoV infections. Written informed consent was obtained from participants and the parents of all children who provided specimens. All samples were anonymized. The production of New Zealand rabbit antiserum was approved by the Animal Care Welfare Committee of National Institute for Viral Control and Prevention, China CDC .MK408529; all accession numbers listed are GenBank accession numbers) isolated from a sporadic case of GII.13 NoV in 2016 in Sichuan, China, (ii) two GII.13 P domains based on the sequences of Goulburn Valley/1983 (DQ379714) and 08N2045/2008 (AB809973), (iii) three GII.21 P domains based on the sequences of OC06060/2006 (AB542916), YO284/2007 (KJ196284), and ZS/2016 (KY407202), (iv) two GII.17 P domains based on the sequences of the outbreak isolates Kawasaki323/2014 (AB983218) and Kawasaki308/2015 (LC037415), and (v) a GII.14 P domain protein GD/2016 (MK850443) isolated from a sporadic case of GII.14 NoV in 2016 in Guangdong, China. The P domain-encoding sequences were chemically synthesized by Genewiz Company . The three GII.13 P domain (SC1065) mutants with 297Y or 361E single mutations, or with the 297Y/361E double mutation, were constructed by overlap PCR using the mutation sites in the corresponding primer pairs.The following P domain proteins were produced and tested in this study: (i) GII.13 P domain protein SC1065/2016 (accession number S-transferase (GST) fusion protein, as described previously . P proteins contained 12 P dimers  to the C-terminal end of the P domain to stabilize P protein formation , while feviously . The P pP dimers .a), Lewis x (Lex), Lewis b (Leb), Lewis y (Ley), type I precursor (Lec), type II precursor (LacNAc), lactose (Lac), sialyl Lea (sLea), sialyl Lex (sLex), Neu5Ac, Neu5Gc, Neu5Acα2-6Gal, and mucin cores 2, 4, and 6 (GlycoTech); text formulas and catalogue numbers are listed in The binding assays of biotin-labeled polyacrylamide (PAA)-conjugated oligosaccharide and P proteins were confirmed by enzyme-linked immunosorbent assay (ELISA), as described previously . The P pNonfat milk and free lactose (Sigma) were used in blocking assays to block glycan binding to Goulburn Valley, 08N2045, and SC1065 GII.13 P proteins, OC06060/2006, YO284/2007, and ZS/2016 GII.21 P proteins, and Kawasaki323/2014 and Kawasaki308/2015 GII.17 P proteins. In the nonfat milk blocking assay, Lec, Lac, or mucin core 2 at 0.2 μg/well was incubated at 4°C overnight with 0%, 2%, 5%, or 10% nonfat milk, while in the lactose blocking assay, Lec, Lac, or mucin core 2 was mixed with 0.625, 1.25., 2.5, 5.0, or 10.0 mg/well of free Lac in PBS. HRP-conjugated streptavidin was added, and the assay was completed as described above .a, Leb, Lex, and Ley phenotypes was used in the saliva binding assay, as described previously  and anti-GII.17 KW323   primary The purified P domain of SC1065 and 08N2045 was concentrated to ∼10 mg/ml. Native crystals of the SC1065 and 08N2045 P proteins were grown using the sitting-drop vapor diffusion method by mixing 1 μl of protein solution with an equal volume of reservoir solution containing 0.2 M sodium citrate tribasic dehydrate, 20% (wt/vol) polyethylene glycol 3350, 0.2 M ammonium acetate, 0.1 M HEPES (pH 7.5), and 25% (wt/vol) polyethylene glycol 3350. SC1065 P protein and Lec disaccharide (Dextra) were mixed at a 1:50 molar ratio and incubated for 5 h at 4°C under the same conditions as the native protein and also like the complex crystals of 08N2045 P domain and core 2 trisaccharide. SC1065N297Y was crystallized under the condition of 8% (vol/vol) Tacsimate (pH 8.0) and 20% (wt/vol) polyethylene glycol 3350. After incubation for 7 days at 18°C, the native and complex crystals were transferred to a cryoprotectant containing mother liquor and 20% (vol/vol) glycerol and subsequently flash-frozen in liquid nitrogen.4RLZ) as a search model. The model was further refined using phenix.refine (https://pymol.org/2/). The representative GII.13 P structures were calculated with the align function in PyMOL.X-ray diffraction data were collected at Shanghai Synchrotron Radiation Facility (SSRF) BL19U and processed with HKL2000. Additional processing was performed using CCP4 software. The structure of the GII.13 SC1065 P domain was determined using the molecular replacement module of PHASER, with the GII.21 P structure  was used to compare the differences between untreated and free-lac-treated groups. In figures, statistical significance is indicated as follows: *, 6JYR, 6JYN, 6JYS, and 6JYO, respectively.The structures of 08N2045, SC1065-lec, 08N2045-Core2, and SC1065-297Y P domains have been deposited under PDB codes


Tight junctions are complex supramolecular entities composed of integral membrane proteins, membrane-associated and soluble cytoplasmic proteins engaging in an intricate and dynamic system of protein–protein interactions. Three-dimensional structures of several tight-junction proteins or their isolated domains have been determined by X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. These structures provide direct insight into molecular interactions that contribute to the formation, integrity, or function of tight junctions. In addition, the known experimental structures have allowed the modeling of ligand-binding events involving tight-junction proteins. Here, we review the published structures of tight-junction proteins. We show that these proteins are composed of a limited set of structural motifs and highlight common types of interactions between tight-junction proteins and their ligands involving these motifs. As most apical elements of the junctional complex, tight junctions (TJs) were distinct by the apparent fusion of adjacent cell membranes over variable distances and appeared as a diffuse band of dense cytoplasmic material in the electron microscope. TJs formed a continuous belt-like structure, whereas desmosomes displayed discontinuous button-like structures, and adherens junctions (AJs) were intermediate in appearance. The molecular composition of TJs was revealed in subsequent work by many laboratories, e.g., XΦ residue .Following the three N-terminal PDZ domains, ZO proteins contain a SH3-GUK module. Crystal structure analysis of the ZO-1 SH3-GUK tandem domain confirmed independent folding of the SH3 and GUK domains, and pulldown assays identified the downstream U6 loop as an intramolecular ligand of the SH3-GUK core with a potential role in regulating TJ assembly in vivo . CrystalZO-1 is distinct from its paralogs ZO-2 and ZO-3 by the presence of an extended C-terminal region harboring a ZU5 domain ,105 and In the MAGI proteins (MAGUKs with inverted domain structure), the characteristic arrangement of PPI domains present in common MAGUK proteins is inverted. Furthermore, the MAGI proteins contain two WW domains in place of the SH3 domain found in MAGUKs  107]. T. T107]. The Crumbs/PALS1/PATJ complex is involved in establishing and maintaining cell polarity and located in the cytoplasmic plaque of TJs ,3,111. CThe PATJ–PALS1 interaction is mediated by the single L27 domain of PATJ and the N-terminal L27 domain (L27N) of PALS1 . A crystDrosophila Crumbs. The 1.5-Å resolution crystal structure revealed that both the FERM-binding motif, as well as the PDZ-binding motif present in the Crumbs C-terminal peptide contribute to the interaction with moesin. Phosphorylation of the Crb-CT by atypical protein kinase C (aPKC) disrupts the Crumbs/moesin association but not the Crumbs/PALS1 interaction. Crumbs may therefore act as aPKC-mediated sensor in epithelial tissues [The intracellular functions of Crumbs3 (CRB3) are mediated by its conserved 37-aa cytoplasmic tail (Crb-CT) and its interaction with PALS1 and the actin-binding protein moesin. The crystal structure of a PALS1 PDZ-SH3-GUK/Crb-CT complex shows that all three domains of PALS1 contribute to Crb-CT binding . A furth tissues . Similar as the Crumbs/PALS1/PATJ complex, the evolutionarily conserved PAR-3/PAR-6/aPKC complex is associated to TJs and crucial for establishing and maintaining cell polarity. The complex formed by the PAR (partition defective)-3 and PAR-6 proteins, as well as the atypical protein kinase C (aPKC) interacts with subunits from the Crumbs/PALS1/PATJ complex and is regulated by binding to the small GTPases CDC42 and RAC1. Composition and stoichiometry of the PAR-3/PAR-6/aPKC complex are linked to cell polarity and to the cell cycle .Drosophila homolog Stardust. The regulation of ligand binding to PAR-6 PDZ by CDC42 has been structurally characterized in a number of studies. A 2.5-Å crystal structure of a PAR-6 PDZ-bound internal dodecapeptide derived from PALS1 revealed a characteristic deformation of the carboxylate-binding loop of PAR-6 PDZ relative to the structure with bound C-terminal ligand  [PAR-3 acts as central organizer of the PAR-3/PAR-6/aPKC complex and is thus essential for establishment and maintenance of cell polarity. In le (JAM) ,123. In le (JAM) . PAR-3 cle (JAM) . The abile (JAM) .The second PDZ domain of PAR-3 binds phosphatidylinositol (PI) lipid membranes with high affinity as shown in a biochemical and NMR study of PAR-3 PDZ2. This study also showed that the lipid phosphatase PTEN (phosphatase and tensin homolog) binds PAR-3 PDZ3 and thus cooperates with PI in regulating cell polarity through PAR-3 . A threeIn addition to the MAGUK proteins, the subunits and regulators of the Crumbs/PALS1/PATJ and PAR-3/PAR-6/aPKC complexes, various other cytoplasmic proteins are associated with TJs. The multiple PDZ domain protein 1  contains 13 PDZ domains. MUPP1 is a paralog of PATJ, shares several TJ-binding partners  and a siCldn4 gene, being under transcriptional control by GRHL2, GRHL3, the androgen receptor, retinoic acid/retinoid X receptors, and p63 in different tissues [The molecular composition of TJs varies significantly between different epithelia and determines their dual functions as effective barriers for solutes or channels for particular classes of solutes . Therefo tissues ,146,147, tissues ,150,151, tissues ,12 will Here, we have reviewed three-dimensional structures of TJ proteins, focusing on the core TJ complex. It becomes clear that our knowledge of these structures is fairly incomplete, because most TJ proteins do not lend themselves easily to structure analysis due to their size and/or the presence of TM regions, natively unfolded polypeptide segments or heterogeneous post-translational modifications ,75,76. O


Penile lymphangiomas are rare manifestations of lymphangiomas or lymphatic malformations which are more commonly found in the head or neck region of the body. Lymphangiomas are further categorized as lymphangioma circumscriptum, cavernous lymphangioma, cystic hygroma, or acquired lymphangiomas , based on their depth and etiology.A literature review revealed only 30 cases of penile lymphangioma between 1947 and March 30, 2018. Several causes were attributed to the acquired penile lymphangiomas, including trauma, phimosis, and infection. While penile lymphangiomas can be initially mistaken for an infection, a thorough history and physical examination is sufficient to clinically diagnose a lymphangioma of the penis. Historically, surgical excision has been the gold standard of treatment for this condition. When asymptomatic, patients may opt for conservative management with avoidance of mechanical trauma alone. Other physicians have revealed novel treatment plans to rid patients of their penile lymphangioma such as a staged laser procedure.In this article, we elucidate the causes, symptoms, treatments, and outcomes associated with penile lymphangiomas found in the literature while also presenting the case of a 30-year-old African-American man diagnosed with acquired penile lymphangioma. Redenbacher et al. first described lymphangiomas, or “lymphatic malformations,” in 1828 . The objWe conducted a literature review identifying all publications using the keywords: “penile” OR “penis” AND “lymphangioma” OR “lymphangiectasis” OR “lymphangiectasia.” Following our institutional protocol, the literature review identified relevant studies via a computer-aided search of American  and European articles . Publications found in languages other than English were excluded. Publications reporting lymphangiomas of the scrotum, perineum, and/or surrounding area not including the penis were excluded. The publications reporting benign transient lymphangiectasis of the penis (BTLP) were also excluded as BTLP, commonly known as sclerosing lymphangitis, is a transient condition that often results after sexual activity and holds minimal medical or surgical relevance. Classifications of penile lymphangioma found in Table , and the patient was referred to urology for evaluation and management. With the urologist, the patient elected for surgical intervention due to cosmetic concerns despite the asymptomatic nature of the lymphangioma.A 30-year-old African-American male presented to his primary care physician with a chief complaint of a several-year history of unhealing wounds on the right side of his penile shaft after his penis was caught in his zipper several years ago. Patient became concerned after noting white penile discharge 2 weeks prior. He denies any anal or oral lesions as well as exposure to any sexually transmitted diseases (STDs). Following outpatient specialty referral, the dermatologist reported excess skin tissue with underlying edema circumferentially on the distal penile shaft with overlying multiple firm skin-colored papules, some with exophytic crusting Fig. . ChlamydThe patient underwent circumcision for redundant prepuce, excision of the skin lesion and penile foreskin reconstruction. A circumferential incision was made on the mucosa 0.5 cm proximal to the glans, distal to the lymphangioma. The foreskin was then retracted and another circumferential incision was made around the mucosal skin. The foreskin was then dissected using Bovie cautery and blunt dissection while the foreskin with lymphangioma tissue was excised. Intraoperative and postoperative courses were unremarkable. At 1-month follow-up, the patient reported no pain, erythema or discharge from the wound.The current literature review identified 27 cases in 25 publications. In the largest group, the 18 acquired lymphangiomas of the penis identified in the literature review encompassed a wide variety of cases. One was attributed to phimosis and 2 to trauma , 5. Two Lymphangioma circumscriptum of the penis in this literature review was reported in eight, five of which were ruled to be congenital. The other three were notable for comorbidities of ulcerative colitis, recurrent cellulitis, and Lichen planus –14. The Lymphangiomas are generally described as uncommon, hamartomatous malformations of the lymphatic system. Based on depth and size of the lymph vessels, classifications include lymphangioma circumscriptum , cavernous lymphangioma and cystic hygroma (most deep). Further subdivisions derive from perceived cause. Congenital lymphangiomas are thought to result from fetal lymph vessels that failed to involute and/or failed to join with the central lymphatic system. On the contrary, acquired lymphangiomas may result from trauma, certain infections , radiotherapy, pregnancy, scleroderma, severe phimosis or STDs .Although the causes of these various types of lymphangioma may differ, the clinical presentation is often similar. The majority of the penile lymphangiomas present as asymptomatic, fluid-filled, translucent lesions or vesicles most commonly on the shaft or coronal sulcus of the penis. Symptomatic lesions, on the other hand, typically focus on sexual dysfunction. One case of acquired lymphangioma presented with severe phimosis and inability to produce an erection . Also, oMany authors may argue that lymphangiomas of the penis do not always require treatment, given the mostly asymptomatic nature of these lesions. However, patients may request intervention for cosmetic reasons. Of the 18 acquired lymphangiomas reported in Table Of the 3 cavernous lymphangiomas reported in Table In summary, penile lymphangioma can be divided into four categories: 1) acquired, 2) lymphangioma circumscriptum, 3) cavernous lymphangioma, and 4) cystic hygroma. Commonly mistaken for infectious lesions, lymphangiomas underscore the importance of an appropriate history and physical to properly identify and treat the lymphatic malformation.


Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5’ external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5’ external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5’ external transcribed spacer ribonucleoprotein – provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly. In yeast, the mature ribosome is a 3.2 MDa ribonucleoprotein complex composed of 79 proteins and four ribosomal RNAs (rRNA) assembled into the small and the large subunit. However, throughout its biogenesis over 200 assembly factors are known to sequentially and transiently associate with pre-ribosomal intermediates that mature as they are trafficked from their origin in the nucleolus through the nucleus to their final cytoplasmic destination . During Ribosome assembly starts with the RNA polymerase-I driven transcription of the 35S precursor ribosomal RNA (pre-rRNA) in the nucleolus. This primary transcript contains rRNAs of the small ribosomal subunit  and the large ribosomal subunit  flanked by external transcribed spacers  and separated by internal transcribed spacers . Assembly factors are co-transcriptionally recruited and associate with the nascent pre-rRNA, resulting in a chronology of biogenesis that starts at the 5’ end, where the 5’ ETS and rRNA for the small subunit reside. The first structurally defined particle that assembles co-transcriptionally is therefore the small subunit processome, a giant precursor of the small subunit .Initial studies showed that the depletion of early assembly factors resulted in reduced SSU processome formation . HoweverBased on genetic data, a hierarchical model was proposed , which wRecent structural studies of the fully assembled SSU processome have shown that the 5’ ETS and its associated multi-protein complexes UtpA, UtpB and U3 snoRNP form a scaffold for the assembly of individual rRNA domains, thus further highlighting the central role of the 5’ ETS .However, since the molecular basis for a hierarchical assembly model of the SSU processome remains elusive, two fundamentally different models can explain the available data:Early assembly factors could provide a direct and hierarchical support for the recruitment of subsequent proteins and RNAs. Alternatively, individual pre-rRNA sequences could independently recruit their assembly factors and may be mutually dependent on each other for the final SSU processome assembly.Here we determine the mechanisms underlying the controlled assembly of the SSU processome by using a combination of biochemical, mass-spectrometry and cryo-electron microscopy (cryo-EM) analyses of nucleolar particles preceding the SSU processome. We show that the 5’ ETS and stable rRNA domains are functionally independent in their ability to recruit their own assembly factors, and the strict order of events is enforced through molecular switches within the 5’ ETS ribonucleoprotein (RNP) that require the presence of all rRNA domains for the final SSU processome to be formed.To determine the order in which ribosome assembly factors associate with pre-rRNA, previous systems employed 3’ end truncations of rRNA mimics to emulate continuing transcription . As a reStrikingly, proteins that were previously found to require the 3’ minor domain of the 18S rRNA for their association with the SSU processome  also reqWe determined cryo-EM structures of the 5’ external transcribed spacer in the context of several nucleolar precursors containing either the 5’ ETS alone (17 Å), the 5’ ETS and 5’ domain (9.5 Å) or a pre-rRNA mimic covering the 5’ ETS, 5’ domain and the central domain 4.3 Å)  and the 18S pre-rRNA (box A and box A’)  and 4a–dThe architecture of the 5’ ETS RNP together with analyses of defined in vivo assembled pre-rRNA particles have shed light on the mechanisms of quality control during the formation of the SSU processome. While we cannot categorically rule out the possibility that the particles isolated in this study are off-pathway intermediates, the following observations suggest that this is not the case. Our previous work demonstrated that pre-rRNA mimics containing MS2 loops that are transcribed by RNA polymerase II can serve as the sole source of ribosomal RNA . FurtherContrary to prior models that suggested a hierarchical assembly of SSU processome factors  or moldiS. cerevisiae strain BY4741 were cloned into a derivative of the pESC_URA vector (Agilent Technologies). Primers used to amplify subsections of the rDNA locus are listed in gal1 promoter and upstream of a CYC terminator  (Defined segments of the rDNA locus of rminator . A modifrminator  fused toistance) .pep4 locus of S. cerevisiae strain BY4741 by homologous recombination to yield Y180, which was used to create all subsequent yeast strains harboring C-terminal streptavidin-binding-peptide (SBP) tags on endogenous ribosome biogenesis factors  (B079 was linearized and integrated into the nd Mrd1) . Homologpep4 locus and a streptavidin binding peptide (SBP)-tagged ribosome assembly factor . The cells were flash frozen in liquid N2 and subsequently cryo-ground using a planetary ball mill (Retsch PM100).Transformed cells were grown on -URA synthetic dropout media plates supplemented with 2% glucose (w/v) and appropriate antibiotics  for 2 da2, 5% glycerol, 0.1% NP40) supplemented with protease inhibitors . Insoluble fractions were pelleted for 20 min at 40’000 x g, 4°C, and the supernatant was incubated with anti-GFP nanobody coupled sepharose (Chromotek) for 3 to 4 hr at 4°C. Pre-rRNA mimics and their associated proteins were eluted by 3C-protease cleavage at 4°C for 1 hr. Subsequently, eluted RNPs were applied to Streptavidin-coupled sepharose resin for 1 hr at 4°C, washed four times with wash buffer , 150 mM NaCl, 5 mM MgCl2) and released from beads by incubation in 50 mM Tris-HCl, pH 7.5 (RT), 150 mM NaCl, 5 mM MgCl2, 5 mM D-biotin for 30 min at 4°C. Eluted samples were either directly used for RNA extraction, mass-spectrometry analysis and negative stain electron microscopy sample preparation, or supplemented with 5 mM putrescine, 1 mM spermidine and 0.03% Triton X-100 for subsequent cryo-electron microscopy studies.10–40 grams of cryo-ground yeast powder were resuspended in binding buffer , 150 mM NaCl, 5 mM MgCl5’-domain), B222 , B223 (5’ ETS-18S3’major-domain) or B224 (5’ ETS-18S3’minor domain) were applied on glow-discharged carbon coated grids . Subsequently, grids were washed three times with ddH2O, twice with 2% (w/v) 0.2 μm-filtered uranyl acetate and air dried. Micrographs were acquired on a Philips CM10 operated at an acceleration voltage of 100 kV equipped with a XR16-ActiveVu (AMT) camera at a nominal magnification of 39,000 and a calibrated pixel size of 3.4 Å at the specimen level.3.5 μl of pre-ribosomal particles purified from Y186 transformed with B117 (5’ ETS), B221  with absorbances of 0.35 mAU , 0.65 mAU , 0.8 mAU  at 260 nm  were supplemented with 5 mM putrescine, 1 mM spermidine and 0.03% Triton X-100. 3.5 μl of sample was applied on glow-discharged lacey-carbon grids containing a thin carbon film . Following a 15 s sample incubation period at close to 100% humidity, grids were blotted for 1.5–2.5 s with a blotting force of 0 and flash frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific).Samples in elution buffer , 150 mM NaCl, 5 mM MglClCryo-EM data collection was performed either on a Talos Arctica or Titan Krios (Thermo Fisher Scientific) operated at 200 kV or 300 kV respectively, both mounted with a K2 Summit detector . SerialEM  was used-/Å2) over a defocus range of 1–3.5 μm at 1.6 Å pixel size (super-resolution pixel size 0.8 Å). Motion correction within RELION-3 (http://www.mrc-lmb.cam.ac.uk/kzhang/) yielding 275’080 particles   was used2 in a UV Stratalinker 2400 (Stratagene).Total cellular RNA was extracted from 0.2 gram of frozen yeast cells after lysis by bead beating in 1 ml Trizol (Ambion). To isolate RNA from purified pre-ribosomal particles, 0.5 ml Trizol (Ambion) was added to the final D-biotin elutions and the extraction was performed according to the manufacturer’s instructions. For the analysis of pre-rRNA processing states by Northern blotting, 3 μg total cellular RNA or ~100 ng of RNA extracted from purified RNPs, were loaded in each lane of a 1.2% agarose formaldehyde-formamide gel and separated at 75V for 2.5 hr. After running, the separated RNA was transferred onto a cationized nylon membrane  using downward capillary transfer and cross-linked to the membrane for Northern blot analysis by UV irradiation at 254 nm with a total exposure of 120 millijoules/cmPrior to the addition of γ−32P-end-labeled DNA oligo nucleotide probes , cross-lPurified RNP samples were dried and dissolved in 8 M urea/0.1 M ammonium bicarbonate/10 mM DTT. After reduction, cysteines were alkylated in 30 mM iodoacetamide. Proteins were digested with LysC  in less than 4 M urea followed by trypsination  in less than 2 M urea. Digestions were halted by adding TFA and digests were desalted  and analS. cerevisiae Uniprot protein database (October 2018) concatenated with the MS2-protein sequence and common contaminations. For the search and quantitation, MaxQuant v. 1.6.0.13 (Data were quantified and searched against the 1.6.0.13  was usedst sample) or technical duplicates (2nd sample), three metabolic enzymes , which we considered to be ‘innocent bystanders’ were examined. The signal for the three proteins were comparable between all samples. Hereafter we used the MS2-protein signal to adjust the iBAQ values for each ribosome biogenesis protein of interest. Data are available in Data analysis: Log2 transformed Label Free Quantitation (LFQ) or intensity Based Absolute Quantitation (iBAQ) values  were useThe cryo-EM density map for the 5’ ETS RNP has been deposited in the EM Data Bank with accession code EMD-0441. Coordinates for the 5’ ETS RNP have been deposited in the Protein Data Bank under accession code 6ND4. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by four peer reviewers and the evaluation has been overseen by Cynthia Wolberger as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: John Woolford (Reviewer #3), Katrin Karbstein (Reviewer #4).Thank you for submitting your article "Conformational switches control early maturation of the eukaryotic small ribosomal subunit" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.I want to stress that all reviewers largely agree on the strengths and shortcomings of the manuscript, and that their comments and concerns are highly overlapping. I hope that you find these comments constructive and helpful in revising this work.Summary:Conformational changes that accompany the earliest stages of ribosome biogenesis are crucial for setting up assembly and maturation of the particles, yet are difficult to observe because the earliest intermediates are short lived. The authors of this manuscript used a clever approach to address this problem by expressing fragments of the pre-rRNA in yeast, which cannot mature. Comparison of the proteins bound to these rRNA fragments (by MS), and the structures (by cryo-EM) of partial assemblies with the contents and structure of the processome (the downstream intermediate) suggest that (i) individual domains can assemble proteins bound to them independently of other domains, that (ii) assembly of the full processome requires the presence of all domains (iii) that UtpB undergoes a major opening during assembly to support the recruitment of late binding factors. Overall, this manuscript is considered of high interest to the field, but major improvements in the presentation and explicit mentioning of the caveats of the system must be provided. In addition, there are some requests for additional experimental data.Essential revisions:Necessary experimental revisions:1) Please analyze the binding of proteins to the 3' domain. In order to make the argument that each domain can bind its interaction partners directly, it was felt that this piece of data was needed.2) Please provide a biological replicate of the mass spectrometry data, as well as a more detailed description of what is meant by "technical" replicates. In addition, stoichiometry of the assembly factors (relative to rRNA or r-proteins) must be addressed.3) Please map the cleavage products in Figure 4—figure supplement 2 to support or refute the claim that these molecules are biologically relevant. (The 5' ETS probe and the A0-A1 show RNA pieces of the length expected for just A1 (no A0) processing for the RNPs containing ending after the 5', central and 3' major domains. For the one ending at the 3' minor domain, there is a piece consistent with A0 cleavage. Further supporting the notion that this is the 5' ETS ending at A0, this latter piece disappears with the A0-A1 probe.) This figure demonstrates A1 cleavage in all molecules but A0 cleavage only after the SSU processome is assembled (and is directly contradicting statements by the authors). This could be reconciled if A1 cleavage is actually cleavage near A1.Necessary revisions in text:1) Caveats must be introduced into the manuscript in multiple instances:a) Whether or not these molecules are functionally relevant cannot be ascertained, and this must be clearly acknowledged. The surprising lower stringency of A1 cleavage relative to A0 cleavage must also be clearly acknowledged as suggesting that these intermediates differ somehow from real physiological intermediates.b) Whether a trans-mechanism for assembly (an endogenous 5' ETS binding to the independent domains to facilitate assembly) could be functional as suggested by a reviewer should be acknowledged or ruled out in the ts PolI strains from Nomura.2) The existing literature must be discussed more thoroughly. This includes work on the assembly of individual domains, as well as changes during assembly visualized by other methods. Furthermore, the literature should be inspected for evidence that some of the factors recruited late are recruited in response to a conformational change .3) The authors should analyze the "class 3" particles or address why they excluded these from their analysis. This is especially important because an eye-ball analysis suggests it might contain UtpB in the "mature" conformation.4) The authors must revise their description of the molecule not being cleaved, taking account of the actual Northern data in Figure 4—figure supplement 2.5) The authors must better describe that the "5' ETS particle" is and if it contains more than just the 5' ETS, as believed, its name must be revised.6) Figure organization and clarity should be improved. Specific comments include:a) For Figure 1, I would suggest turning the RNA around (from yellow to black), as we are used to read from top to bottom. The same must happen for Figure 1B, and in panel B I would also suggest that the authors include the data from their previous work on the 5' ETS, and then go in the following order 5' ETS, 5' domain, central domain, etc. This would be the order of assembly from right to left. In Figure 1C, I think the authors should have the structures from Figure 1—figure supplement 2A-C, aligned with the rest of the figure.b) Several reviewers comment on Figure 2 being very busy. I would suggest having the 5' ETS left and the SSU processome right, and then keep that throughout the figure. It was suggested to zoom in independently to each of the late factor binding sites. E.g., in Figure 2B it is unclear where Rrp5 is, and how that changes. Nop14 is not in the figure but referred to. Changes in the Noc4 binding site are unclear. Critically, in Figure 2C it is not clearly visible that A0 and A1 are uncut. That cleavage site needs to be pointed out.the major conclusion of the work. I would also encourage the authors to turn the presentation in the manuscript around: it is closed in the 5' ETS RNP and opens up. This is (they think) the order in which assembly happens.c) The data currently presented in Supplementary Figure 6 need to be in the main paper. This is a major conclusion, perhaps d) The legend for Figure 4 needs to stress that these are inferred not observed. It should be added where these inferences are coming from , and it needs to be clarified in the legend that some AFs leave the complex according to this model.e) Figure 4—figure supplement 1C: Can the authors label the protein bands in the gels?eLife. Your revised article has been favorably evaluated by Cynthia Wolberger (Senior Editor), a Reviewing Editor, and two reviewers.Thank you for resubmitting your work entitled "Conformational switches control early maturation of the eukaryotic small ribosomal subunit" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:each rRNA domain of the small ribosomal subunit can independently recruit its own biogenesis factors in vivo." Similar statements are found in the Introduction and Discussion.There is no concern that you were unable to assemble just the 3' domain. But there is unanimous agreement that this requires you to qualify some of your statements, including in the Abstract, which currently states: "Here we show that All reviewers are also unanimous that a qualifying sentence about the functionality would be "scholarly", given that you cannot show it due to the limitations of the assay. This could be along the lines of: "While we cannot rule out the possibility that these are off-pathway intermediates, the following strongly suggests that this is not the case." You do not need to use that wording, but you get the idea. I also want to make it clear that all reviewers specifically state that they do not feel that this affects the impact of the work.There is also unanimous agreement that the wording 5' ETS particle is inappropriate. In the 2015 paper that you reference you actually did use a 5' ETS RNA and found 26 proteins assemble. Nonetheless in here you are studying the structure of an RNP containing 5' ETS, and the 5' and central domains of 18S rRNA. Thus, you should designate the molecule accordingly. I think the confusion comes from the fact that you refer to 5' ETS -particle as a substructure, while the writing as well as the wording particle implies a molecule.Examples include: "We determined the cryo-EM structures of the 5' ETS (delete particle) in the context of […]", then later in the same paragraph: "[…], we observed the 5' ETS substructure/RNP as the unifying and stable structural entity […].""Strikingly the 5' ETS substructure/ RNP etc… adopts a structure […]".I am sure there are more. All four reviewers, all experts in the field, did not understand which molecule you are studying, or that you refer to a changing substructure. That suggests the writing is unclear.Finally, in Figure 1, it's currently impossible to distinguish between missing values and measurements of low abundance as they are both white – this should be changed so that the figure can be interpreted. Essential revisions:Necessary experimental revisions:1) Please analyze the binding of proteins to the 3' domain. In order to make the argument that each domain can bind its interaction partners directly, it was felt that this piece of data was needed.“While the 5’ domain and central domain could be stably expressed in isolation, the 3’ major domain was unstable when expressed individually, which prevented a direct analysis of bound factors .”We thank the reviewers for this suggestion. Similar to the other domains , we have expressed the 3’ major domain with MS2 aptamers at the 3’ end. However, in contrast to the 5’ and central domain, this construct in isolation is not stable when expressed in yeast as northern blotting of total cellular RNA showed that only the MS2 aptamers remain in yeast cells . The degradation of this construct in vivotherefore prohibits the analysis of associated proteins. To clarify this point, we also added the following statement to the Results (subsection “18S rRNA domains independently recruit their assembly factors”): 2) Please provide a biological replicate of the mass spectrometry data, as well as a more detailed description of what is meant by "technical" replicates. In addition, stoichiometry of the assembly factors (relative to rRNA or r-proteins) must be addressed.For biological replicates, we have re-expressed all rRNA constructs used for Figure 1B in yeast. Subsequently we have performed two independent RNA-protein purifications for mass spectrometry analysis for each of these constructs, which are technical replicates as they originate from the same biological source. We have combined all the data in the revised version of Figure 1B, which now contains mass spectrometry data for each construct in quintuplets; three technical replicates of the first biological duplicate  and two technical replicates of the second biological duplicate (as requested by the reviewers).We note that the determination of an accurate stoichiometry between ribosomal proteins, ribosome assembly factors and RNAs is not possible with this approach. This is evidenced by the fact that any attempts of determining accurate stoichiometries in previous studies using this method  have been unsuccessful when comparing these data to subsequent structural data on the small subunit processome  and large subunit precursors .3) Please map the cleavage products in Figure 4—figure supplement 2 to support or refute the claim that these molecules are biologically relevant. (The 5' ETS probe and the A0-A1 show RNA pieces of the length expected for just A1 (no A0) processing for the RNPs containing ending after the 5', central and 3' major domains. For the one ending at the 3' minor domain, there is a piece consistent with A0 cleavage. Further supporting the notion that this is the 5' ETS ending at A0, this latter piece disappears with the A0-A1 probe.) This figure demonstrates A1 cleavage in all molecules but A0 cleavage only after the SSU processome is assembled (and is directly contradicting statements by the authors). This could be reconciled if A1 cleavage is actually cleavage near A1.In our original submission a northern blot with an MS2 probe of total cellular RNA highlighted that in vivoeach of the constructs was present with the expected length as the dominant species . We also showed that specifically upon purification of these particles, degradation products appear in the northern blots when the MS2, 5’ ETS or A0-A1 probes are used . This suggested that degradation occurs during the purification of these particles. The size of the major degradation product above 750 nucleotides further suggested non-specific cleavage events since a site-specific cleavage at site A1 would generate a product of exactly 700 nucleotides for the 5’ ETS and A0-A1 probes.central domain), which showed the most prominent degradation product with the 5’ ETS probe .To further exclude the possibility that A1 cleavage occurs within these constructs, we have used the construct terminating with the central domain  with the previous probes  as well as a probe that binds immediately downstream of the A1 cleavage site (A1 probe) within the 5’ domain. As shown in the new supplementary figure , the major degradation product, which runs above 750 nucleotides is detected by the 5’ ETS, A0-A1 and A1 probes. This proves two important points:In a new supplementary figure  we have probed the representative construct (5’ ETS-18S1) Non-specific cleavage, and not A1 cleavage, is responsible for the observed degradation product. This most likely occurs during the purification.2) The pre-ribosomal particles generated by our method are therefore biologically relevant since A1 cleavage would not be expected.5’ domain; 5’ ETS-18Scentral domain; 5’ETS-18S3’ major domain). Second, for the onlyconstruct that can form a small subunit processome (5’ETS-18S3’ minor domain) we observe A0 cleavage , which is expected based on our previous detailed characterization of the pre-ribosomal RNAs present in the small subunit processome .These data are further consistent with our model since early particles that are not competent to form the SSU processome do not undergo A0 or A1 cleavage whereas A0 cleavage is required for SSU processome formation to occur. First, our data conclusively rule out A1 cleavage for any of the incomplete pre-ribosomal RNA mimics  Caveats must be introduced into the manuscript in multiple instances:a) Whether or not these molecules are functionally relevant cannot be ascertained, and this must be clearly acknowledged. The surprising lower stringency of A1 cleavage relative to A0 cleavage must also be clearly acknowledged as suggesting that these intermediates differ somehow from real physiological intermediates.We believe that the new supplementary figure  has addressed the concerns raised in point 3 where additional experiments were requested. In summary, the additional experiments showed that no A1 cleavage occurs in these constructs until the SSU processome is formed. We therefore believe that these preribosomal RNAs are indeed functionally relevant.b) Whether a trans-mechanism for assembly (an endogenous 5' ETS binding to the independent domains to facilitate assembly) could be functional as suggested by a reviewer should be acknowledged or ruled out in the ts PolI strains from Nomura.We believe that a trans-mechanism is highly unlikely for several reasons.First, small subunit processome formation is an intramolecular reaction as far as preribosomal RNA and its associated factors are concerned.Second, within the small subunit processome the proximity of additional assembly factors  and U3 snoRNA, which have multiple interactions with different domains of the same pre-ribosomal RNA, further favor an intramolecular reaction as their high local concentrations will easily outcompete assembly factors that could interact in trans.Third, if there was a trans-mechanism, an evolutionary separation of the 5’ ETS and the 18S rRNA coding region would be expected as each sequence could exist in isolation. However, instead the 5’ ETS always directly precedes the 18S rRNA coding region.Fourth, if a 5’ ETS particle could assemble other ribosomal RNA domains in trans, we would expect to have seen mass spectrometry evidence of these interactions in the form of 5’ ETS associated factors in the pulldowns for just the 5’ or central domains. However, this is not the case .In light of the four points outlined above we do not think it is appropriate to mention a trans-mechanism in this manuscript.2) The existing literature must be discussed more thoroughly. This includes work on the assembly of individual domains, as well as changes during assembly visualized by other methods. Furthermore, the literature should be inspected for evidence that some of the factors recruited late are recruited in response to a conformational change .We thank the reviewers for this comment and have expanded the Introduction to include prior literature on individual domains and their associated factors. In particular we have highlighted the use of Miller spreads, which we believe is highly relevant for this manuscript and the proposed model.Regarding assembly factor recruitment in response to a conformational change, previous work from our laboratory as well as that of Keqiong Ye’s group has shown that the addition of 167 nt of the 3’ minor domain is sufficient for the recruitment of a large number of factors to small subunit processomes . Both laboratories postulated that the addition of the 3’ minor domain is not a direct binding site for these factors and this was further confirmed by all structures of small subunit processomes showing that these factors bind on the outside of the small subunit processome and not the 3' minor domain directly . This observation can only be rationalized by the creation of new binding sites for these factors, which previously did not exist as the individual domains of the SSU processome  were physically separated from one another. The structural changes of UtpB first described in this manuscript lend additional support to this model.3) The authors should analyze the "class 3" particles or address why they excluded these from their analysis. This is especially important because an eye-ball analysis suggests it might contain UtpB in the "mature" conformation.To analyze the state of UtpB within class 3, this class was subjected to a subsequent 3D classification that revealed that only 22% (less than 5% of the entire dataset) contain the isolated tetramerization domain of UtpB in a mature configuration. The small number of particles and limiting resolution of the resulting reconstruction prevented further analysis. Since 64% of all particles contain the compacted configuration of UtpB, which we also observe in the reconstructions obtained for just the 5’ ETS as well as the 5’ ETS -5’ domain construct, we believe that this dominant state is physiologically relevant.4) The authors must revise their description of the molecule not being cleaved, taking account of the actual Northern data in Figure 4—figure supplement 2.The actual Northern data  show that cleavage at site A1 has not occurred.5) The authors must better describe that the "5' ETS particle" is, and if it contains more than just the 5' ETS, as believed, its name must be revised.Originally, we coined the term “5’ ETS particle” for a particle that contains the 5’ external transcribed spacer and its associated 26 proteins . Since our visualized 3D reconstruction contains precisely these components, we believe that the term is appropriate. To clarify this point in the manuscript, we have added a sentence in the Introduction, which states:“The particle containing the 5’ ETS region and its associated 26 unique polypeptides was termed the 5’ ETS particle .”6) Figure organization and clarity should be improved. Specific comments include:a) For Figure 1, I would suggest turning the RNA around (from yellow to black), as we are used to read from top to bottom. The same must happen for Figure 1B, and in panel B I would also suggest that the authors include the data from their previous work on the 5' ETS, and then go in the following order 5' ETS, 5' domain, central domain, etc. This would be the order of assembly from right to left. In Figure 1C, I think the authors should have the structures from Figure 1—figure supplement 2A-C, aligned with the rest of the figure.While we thank the reviewer for this suggestion, we strongly believe that Figure 1 should remain exactly as it is for the following reasons:1) Turning panels A and B would not scientifically change the interpretation of this data.2) We believe that including data from previously published primary literature in a new publication is problematic.3) As only the highest resolution reconstruction is discussed in this manuscript, including other reconstructions would not help in clarifying the content of this manuscript for the readership.b) Several reviewers comment on Figure 2 being very busy. I would suggest having the 5' ETS left and the SSU processome right, and then keep that throughout the figure. It was suggested to zoom in independently to each of the late factor binding sites. E.g., in Figure 2B it is unclear where Rrp5 is, and how that changes. Nop14 is not in the figure but referred to. Changes in the Noc4 binding site are unclear. Critically, in Figure 2C it is not clearly visible that A0 and A1 are uncut. That cleavage site needs to be pointed out.We thank the reviewers for this suggestion and agree that Figure 2 in its previous version was quite complex. To simplify this figure , we have split it into two parts. The first part  describes the consequences of conformational changes within the SSU processome and 5’ ETS particle whereas the second part  describes the differences in RNA between the two particles.To clarify the depiction of assembly factor binding sites on UtpB, we have included a visualization that shows these factors as cartoon representations on the surfaces of UtpB  whereas the bottom part of the same panel shows the same structures with the mapped binding sites that are labelled in italics for each of the assembly factors. Nop14 is shown in Figure 3A.Changes in the Noc4 binding site have been re-rendered for clarification in Figure 3E and F.To increase clarity, grey boxes have been removed and the outline width of the rendered structures has been increased.In the RNA description , sites A0 and A1 have been more clearly indicated as requested.c) The data currently presented in Supplementary Figure 6 need to be in the main paper. This is a major conclusion, perhaps the major conclusion of the work. I would also encourage the authors to turn the presentation in the manuscript around: it is closed in the 5' ETS RNP and opens up. This is (they think) the order in which assembly happens.We thank the reviewer for this suggestion. We have accordingly shifted the previous Supplementary Figure 6 into the main text  to put additional emphasis on this point.d) The legend for Figure 4 needs to stress that these are inferred not observed. It should be added where these inferences are coming from , and it needs to be clarified in the legend that some AFs leave the complex according to this model.We have revised the figure and expanded the figure legend of what is now Figure 6 to clarify the above points.e) Figure 4—figure supplement 1C: Can the authors label the protein bands in the gels?Since this manuscript discusses more than 50 assembly factors, the sheer number of these proteins does not allow us to assign bands in each gel, which is why we have used in solution mass spectrometry instead.There is no concern that you were unable to assemble just the 3' domain. But there is unanimous agreement that this requires you to qualify some of your statements, including in the Abstract, which currently states: "Here we show that each rRNA domain of the small ribosomal subunit can independently recruit its own biogenesis factors in vivo." Similar statements are found in the Introduction and Discussion.All reviewers are also unanimous that a qualifying sentence about the functionality would be "scholarly", given that you cannot show it due to the limitations of the assay. This could be along the lines of: "While we cannot rule out the possibility that these are off-pathway intermediates, the following strongly suggests that this is not the case." You do not need to use that wording, but you get the idea. I also want to make it clear that all reviewers specifically state that they do not feel that this affects the impact of the work.There is also unanimous agreement that the wording 5' ETS particle is inappropriate. In the 2015 paper that you reference you actually did use a 5' ETS RNA and found 26 proteins assemble. Nonetheless in here you are studying the structure of an RNP containing 5' ETS, and the 5- and central domains of 18S rRNA. Thus, you should designate the molecule accordingly. I think the confusion comes from the fact that you refer to 5' ETS particle as a substructure, while the writing as well as the wording particle implies a molecule.Examples include: "We determined the cryo-EM structures of the 5' ETS (delete particle) in the context of […]", then later in the same paragraph: "[…], we observed the 5' ETS substructure/RNP as the unifying and stable structural entity […].""Strikingly the 5' ETS substructure/ RNP etc… adopts a structure […]".I am sure there are more. All four reviewers, all experts in the field, did not understand which molecule you are studying, or that you refer to a changing substructure. That suggests the writing is unclear.Finally, in Figure 1, it's currently impossible to distinguish between missing values and measurements of low abundance as they are both white – this should be changed so that the figure can be interpreted.We have addressed the last concerns as outlined below.stable rRNA domains, in other words the 5’ and central domain of the small subunit rRNA but not the 3’ major domain, can recruit their own assembly factors.1) We have specified in the manuscript  that 2) By using the provided template, we have made the following statement regarding the functionality of the studied particles:“While we cannot categorically rule out the possibility that the particles isolated in this study are off-pathway intermediates, the following observations suggest that this is not the case. Our previous work demonstrated that pre-rRNA mimics containing MS2 loops that are transcribed by RNA polymerase II can serve as the sole source of ribosomal RNA. Furthermore, in good agreement with prior genetic and biochemical data, we detected all expected ribosome assembly factors that were associated with the studied pre-rRNA mimics. Lastly, the specific A0 cleavage observed upon SSU processome formation indicates that the studied particles undergo specific quality control as expected for native particles.”3) Thank you for the suggesting “5’ ETS RNP”. We have used this term in all instances where previous terminology was ambiguous.4) We have revised Figure 1 such that proteins that were not detected are now highlighted in light brown.


Cdk5 in the suprachiasmatic nuclei (SCN), the main coordinator site of the mammalian circadian system, shortened the free-running period in mice. CDK5 phosphorylated PER2 at serine residue 394 (S394) in a diurnal fashion. This phosphorylation facilitated interaction with Cryptochrome 1 (CRY1) and nuclear entry of the PER2-CRY1 complex. Taken together, we found that CDK5 drives nuclear entry of PER2, which is critical for establishing an adequate circadian period of the molecular circadian cycle. Of note is that CDK5 may not exclusively phosphorylate PER2, but in addition may regulate other proteins that are involved in the clock mechanism. Taken together, it appears that CDK5 is critically involved in the regulation of the circadian clock and may represent a link to various diseases affected by a derailed circadian clock.Circadian oscillations emerge from transcriptional and post-translational feedback loops. An important step in generating rhythmicity is the translocation of clock components into the nucleus, which is regulated in many cases by kinases. In mammals, the kinase promoting the nuclear import of the key clock component Period 2 (PER2) is unknown. Here, we show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock involving phosphorylation of PER2. Knock-down of  Anyone who has crossed multiple time zones on a long flight will be familiar with jet lag, and that feeling of wanting to sleep at lunchtime and eat in the middle of the night. Many bodily processes, including appetite and wakefulness, roughly follow a 24-hour cycle. These cycles are known as circadian rhythms, from the Latin ‘circa diem’ meaning about a day. An area of the brain called the suprachiasmatic nucleus (SCN) coordinates circadian rhythms. It acts as a master clock by generating a 24-hour signal for the rest of the body to follow. Jet lag occurs when this internal circadian rhythm becomes out of sync with the local day-night cycle.Although jet lag can be uncomfortable, it tends to disappear over the course of a few days. This is because exposure to daylight in our new location resets the SCN master clock, enabling us to adapt to a new time zone. But evidence suggests that long-term disruption of circadian rhythms, for example as a result of shift work, may have lasting harmful effects. These include an increased risk of degenerative brain disorders such as Parkinson's disease and Alzheimer's disease.Brenna et al. now identify a molecular mechanism that could explain this link. A key component of the SCN master clock is a protein called Period2 (PER2). Levels of PER2 rise and fall over each 24-hour period, helping the brain keep track of time. Brenna et al. show that PER2 interacts with CDK5, a protein that helps regulate brain development and that has been implicated in Parkinson's disease and Alzheimer's disease. Reducing CDK5 levels in mice shortened their circadian rhythms by several hours. It also altered the animals’ behavioral patterns over a 24-hour period. Deleting the gene for PER2 had a similar effect, suggesting that CDK5 helps regulate PER2.Future studies should investigate the molecular links between CDK5, circadian rhythms and processes such as neurodegeneration. The results would provide clues to whether manipulating the circadian clock could help prevent or treat neurological disorders. The circadian clock, prevalent in most organisms, is an evolutionary adaptation to the daily light-dark cycle generated by the sun and the earth’s rotation around its own axis . This clDrosophila having various mutations in their period (per) gene  gene . Furthercillator . Every dDrosophila circadian cycle and to adjust its phase to the external light-dark rhythm  phosphorylates per to destabilize it and to prevent its transport into the nucleus . On the k rhythm .Drosophila, with some modifications (Drosophila tim to escort per into the nucleus was replaced by the Cryptochromes (Cry) in the mammalian system , which is the Drosophila dbt orthologue  was found to target CLOCK . CDK5 isPer2 and PHO85, which encodes a cyclin-dependent protein kinase that is orthologous to CDK5 in mammals. Subsequent experiments in mice demonstrated that silencing of Cdk5 in the SCN shortened the clock period. Our study identified CDK5 as a critical protein kinase in the regulation of the circadian clock and in particular as an important regulator of the crucial clock component PER2.In this study, we wanted to identify proteins promoting the nuclear transport of PER2 with focus on kinase(s) acting similarly to shg. Using a genetic synthetic lethal dosage screen in yeast, we observed a genetic interaction between Per2 in budding yeast by using a variation of the Synthetic Genetic Array (SGA) method  method . To this) method , but may) method . Of note) method . Our sead pho85∆ . Among tThe protein kinase CDK5 is mostly expressed in the brain and has previously been implicated in phosphorylation of mammalian CLOCK . HoweverCdk5 in NIH 3T3 fibroblast cells  but comparable to animals with a deletion mutation in the clock gene Per2, designated as Brdm1Per2  in order to label nuclei, with GFP antibody (green) in order to show cells infected by the virus, and with CDK5 antibody (red) in order to compare protein accumulation between the two strains. Scramble as well as shCdk5 mice expressed GFP in the SCN, indicating that the two different viruses infected cells in this brain region (Cdk5 was efficiently suppressed in the SCN by the shCdk5 but not by the scrambled shRNA (Cdk5. The Cdk5 shRNAs was expressed in the SCN (the injection site) and to some extent also dorsal to the SCN but not in distant brain regions (i.e. the piriform cortex) as confirmed by lack of the GFP signal outside of the targeted region  and immunostained with anti-PER2 (red) SCN sections obtained from control, shCdk5 and Brdm1Per2 mice perfused at ZT12. PER2 was observed in the SCN of scramble controls, but was strongly reduced in shCdk5 and almost absent in Brdm1Per2 animals . Addition of roscovitine abolished phosphorylation of PER2 whereas LiCl had no effect (32P panel).In order to understand whether CDK5 phosphorylates the PER2 protein we overexpressed the N-terminal and C-terminal parts of PER2 fused to GST in bacteria  and perfo effect . InteresBrdm1Per2 mice  in the N-terminal part of PER2 using the recombinant protein, which was phosphorylated by CDK5/p35 in vitro. Mass spectrometry revealed several phosphorylation sites at serine and threonine residues, respectively . One of dm1 mice . This sudm1 mice , confirmdm1 mice –6. With dm1 mice , confirmIn order to determine whether PER2 phosphorylation at S394 is time of day-dependent, we collected SCN tissue every 4 hr. The P-S394-PER2 specific antibody detected highest phosphorylation at ZT12 with weaker or no phosphorylation at other time points indicating that S394 is phosphorylated in a time of day-dependent manner . FractioCdk5 in NIH 3T3 cells using the CRISPR/Cas9 method   , indicat of CDK5 , indicat of CDK5 , support of CDK5 , but not of CDK5 .It has been described that nuclear entry of PER2 involves CRY1 . In addiCdk5 strongly and significantly compromised growth  regulate the cell cycle, with few exceptions such as the cyclin dependent kinase 5 (CDK5). This kinase is ubiquitously expressed and its function is vital in post-mitotic neurons, where other CDKs are not active. Although CDK5 is not implicated in cell cycle progression, it can aberrantly activate components of the cell cycle when it is dysregulated in post-mitotic neurons, leading to cell death . Interesd growth .Cdk5 are perinatal lethal   . The mosARPP-32) . Most liARPP-32)  and CDK5ARPP-32)  potentiaPer2 gene  is very similar in PER1 (PFDHSPIR). If PER1 would be phosphorylated by CDK5 at this site at the same rate as PER2, then PER1 as well as PER2 should be absent in the nucleus of SCN cells. This would correspond to a PER1/PER2 double knock-out, which become immediately arrhythmic when subjected to constant darkness  with a stainless-steel wire lid (Techniplast 1264C116), kept in light- and soundproof ventilated chambers. All mice were entrained to a 12:12 hr LD cycle, and the time of day was expressed as Zeitgeber time . Two- to four-month-old males were used for the experiments. Housing as well as experimental procedures were performed in accordance with the guidelines of the Schweizer Tierschutzgesetz and the declaration of Helsinki. The state veterinarian of the Canton of Fribourg approved the protocol. The floxed Per2 mice  are avaimfa1Δ::MFA1pr-HIS3, can1Δ, his3Δ1, leu2Δ0, ura3∆0, lys2Δ0) carrying the plasmid YCplF2-mPer2  was inoculated into 50 mL glucose-containing synthetic dropout medium lacking leucine (SD-Leu) and grown at 30°C overnight with shaking. Cells were then centrifuged, resuspended in 20 mL of the supernatant, poured into a sterile rectangular petri dish, spotted in a 96-well format on rectangular SD-Leu plates (coined ‘bait plates’ hereafter) using a Biomek 2000 robot , and then grown at 30°C for 3 days. In parallel, the gene deletion array in the strain BY4741  was spotted from the storage plates onto fresh G418-containing YPD plates (96-well format) and also grown at 30°C for 3 days. For the mating procedure (overnight at 30°C), colonies from bait plates were  spotted onto plates containing YPD (plus adenine) and the colonies from the gene deletion array plates were (each separately and in duplicate) spotted on top of them. The next day, the colonies were transferred to G418-containing SD plates lacking lysine, methionine, and leucine (SD-Lys/Met/Leu/+G418) to select for diploids that harbour the YCplF2-mPer2 plasmid. Diploids were then spotted onto plates containing sporulation medium  and incubated at 24°C. After 9 days, tetrads were observed and the colonies were transferred to canavanine-containing SD plates lacking arginine and histidine (SD-Arg/His/+canavanine) to select for MATa haploids. Following growth at 30°C for three days, a second haploid selection was carried out by spotting the colonies on SD-Arg/His/Leu/+canavanine plates (to select for MATa haploids containing the YCplF2-mPer2 plasmid). Following growth at 30°C for 2 days, a third haploid selection was carried out by spotting cells on SD-Arg/His/Leu/+canavanine/+G418 plates (to select for MATa haploids containing the YCplF2-mPer2 plasmid as well as the respective gene deletions of the yeast knockout collection). Following incubation at 30°C for 5 days, colonies were then spotted in parallel onto SD-Arg/His/Leu/+G418 plates and on SD-Raf/Gal-Arg/His/Leu/+G418 plates  to induce expression of PER2. Both types of plates were incubated at 30°C for 4 days and photographed every day. Strains that grew significantly less well on SD-Raf/Gal-Arg/His/Leu/+G418 than on SD-Arg/His/Leu/+G418 included eap1∆, gnd1∆, and pho85∆. In control experiments, the respective original yeast knockout collection mutants were transformed in parallel with the YCplF2-mPer2 or the empty YCplF2 plasmid (−1) instead of ammonium sulfate as nitrogen source, as recommended in The SDL screen was essentially performed as described earlier . Briefly plasmid , selecte plasmid . Please Cdk5. ssAAV-9/2-hSyn1-chI[1x(shNS)]-EGFP-WPRE-SV40p(A) was the scrambled control.Adeno Associate Viruses (AAVs) were produced in the Viral Vector Facility (ETH Zurich). Plasmids used for the production are available on the VVF web site. Two constructs were produced. ssAAV-9/2-hSyn1-chI[mouse(shCdk5)]-EGFP-WPRE-SV40p(A) carried the shRNA against Cdk5 shD, see  which knhttp://products.narishige-group.com/group1/MO-10/electro/english.html) at a rate of 40 nL/min through a pulled glass pipette . The pipette was first raised 0.1 mm to allow spread of the AAVs, and later withdrawn 5 min after the end of the injection. After surgery, mice were allowed to recover for 2 weeks and entrained to LD 12:12 prior to behavior and molecular investigations.Stereotaxic injections were performed on 8-week-old mice under isofluorene anaesthesia using a stereotaxic apparatus (Stoelting). The brain was exposed by craniotomy and the Bregma was used as reference point for all coordinates. AAVs were injected bilaterally into the SCN (Bregma: anterior-posterior (AP) − 0.40 mm; medial-lateral (ML) ±0.00 mm; dorsal-ventral (DV) – 5.5 mm, angle + /- 3°) using a hydraulic manipulator  using a cryostat. Sections chosen for staining were placed in 24-well plates (two sections per well), washed three times with 1x TBS (0.1 M Tris/0.15 M NaCl) and 2x SSC (0.3 M NaCl/0.03 M tri-Na-citrate pH 7). Antigen retrieval was performed with 50% formamide/2x SSC by heating to 65°C for 50 min. Then, sections were washed twice in 2x SSC and three times in 1x TBS pH 7.5, before blocking them for 1.5 hr in 10% fetal bovine serum (Gibco)/0.1% Triton X-100/1x TBS at RT. After the blocking, the primary antibodies, rabbit anti-PER2-1 1:200 , mouse anti-Cdk5 clone 2H6 1:20 , and rabbit anti-GFP 1:500 (abcam ab6556) diluted in 1% FBS/0.1% Triton X-100/1x TBS, were added to the sections and incubated overnight at 4°C. The next day, sections were washed with 1x TBS and incubated with the appropriate fluorescent secondary antibodies diluted 1:500 in 1% FBS/0.1% Triton X-100/1x TBS for 3 hr at RT. (Alexa Fluor 488-AffiniPure Donkey Anti-Rabbit IgG (H+L) no. 711–545–152, Lot: 132876, Alexa Fluor647-AffiniPure Donkey Anti-Mouse IgG (H+L) no. 715–605–150, Lot: 131725, Alexa Fluor647-AffiniPure Donkey Anti-Rabbit IgG (H+L) no. 711–602–152, Lot: 136317 and all from Jackson Immuno Research). Tissue sections were stained with DAPI  for 15 min. Finally, the tissue sections were washed again twice in 1x TBS and mounted on glass microscope slides. Fluorescent images were taken by using a confocal microscope (Leica TCS SP5), and images were taken with a magnification of 40x or 63x. Images were processed with the Leica Application Suite Advanced Fluorescence 2.7.3.9723 according to the study by Immunostained sections were quantified using ImageJ version 1.49. Background was subtracted and the detected signal was divided by the area of measurement. An average value obtained from three independent areas for every section was used. The signal coming from sections obtained from silenced mice was quantified as relative amount to the scramble, which was set to 1. Statistical analysis was performed on three animals per treatment.NIH3T3 mouse fibroblast cells (ATCCRCRL-1658) were maintained in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal calf serum (FCS) and 100 U/mL penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Cdk5 KO cells were produced applying the CRISPR/Cas9 technique according to the manufacturer’s protocol of the company .The following plasmids used were previously described: pSCT-1, pSCT-1mPer2, pSCT-1 mPer-V5, pSCT1 ΔPasA mPer2 -V5, pSCT1 ΔPasB mPer2 -V5  Schmutz. pSCT-1 NIH 3T3 cells were transfected in 10 cm dishes at about 70% of their total confluency using linear polyethylenimine . The amounts of expression vectors were adjusted to yield comparable levels of expressed protein. Thirty hours after transfection, protein extracts were prepared. With regard to immunoprecipitation, each antibody mentioned in the paper was used in the conditions indicated by the respective manufacturer. The next day, samples were captured with 50 µL at 50% (w/v) of protein-A agarose beads (Roche) at 50% (w/v) and the reaction was kept at 4°C for 3 hr on a rotary shaker. Prior to use, beads were washed three times with the appropriate protein buffer and resuspended in the same buffer (50% w/v). The beads were collected by centrifugation and washed three times with NP-40 buffer . After the final wash, beads were resuspendend in 2% SDS, 10% glycerol, 63 mM Trish-HCL pH 6.8 and proteins were eluted for 15 min at RT. Laemmli buffer was finally added, samples were boiled for 5 min at 95° C and finally loaded onto 10% SDS-PAGE gels .2HPO4 × 12 H2O, 2.68 mM KCl, 1.47 mM KH2PO4). Then PBS was added again and plates were kept 5 min at 37°C. NHI3T3 or HEK cells were detached and collected in tubes and washed two times with 1x PBS. After the last washing, pellets were frozen in liquid N2, resuspended in Ripa buffer  with freshly added protease or phosphatase inhibitors, and homogenized by using a pellet pestle. After that samples were centrifuged for 15 min at 16,100 g at 4°C. The supernatant was collected in new tubes and pellet discarded.Medium was aspirated from cell plates, which were washed two times with 1x PBS  and homogenized by using a pellet pestle. Subsequently, samples were kept on ice for 30 min and vortexed five times for 30 s each time. The samples were centrifuged for 20 min at 12,000 rpm at 4°C. The supernatant was collected in new tubes and the pellet discarded.Total brain or isolated SCN tissue was frozen in liquid NTissues or cells were resuspended in 100 mM Tris-HCl pH 8.8/10 mM DTT and homogenized with a disposable pellet pestle. After 10 min incubation on ice, the samples were centrifuged at 2500 g for 2 min at 4°C and the supernatant discarded. After adding 90 μL of completed cytoplasmic lysis buffer , NaF, PMSF, ß-glycerophosphate), the pellet was resuspended by vortexing, followed by centrifugation at 5200 rpm for 2 min at 4°C. The supernatant transferred into a fresh 1.5 mL tube was the CYTOPLASMIC EXTRACT. The pellet was washed three times with cytoplasmic lysis buffer and resuspended in 45 μL 1x NDB  containing 2x proteinase and phosphatase inhibitors followed by adding 1 vol of 2x NUN . After vortexing the samples were incubated 30 min on ice, centrifuged 30 min at 13,000 rpm at 4°C and the supernatant that was transferred into a fresh tube was the NUCLEAR EXTRACT.A protein amount corresponding to between 400 and 800 µg of total extract was diluted with the appropriate protein lysis buffer in a final volume of 250 µL and immunoprecipitated using the indicated antibody (ratio 1:50) and the reaction was kept at 4°C overnight on a rotary shaker. The day after, samples were captured with 50 µL of 50% (w/v) protein-A agarose beads (Roche) and the reaction was kept at 4°C for 3 hr on a rotary shaker. Prior to use, beads were washed three times with the appropriate protein buffer and resuspended in the same buffer (50% w/v). The beads were collected by centrifugation and washed three times with NP-40 buffer . After the final wash, beads were resuspendend in 2% SDS 10%, glycerol, 63 mM Trish-HCL pH 6.8 and proteins were eluted for 15 min at RT. Laemmli buffer was finally added, samples were boiled 5 min at 95° C and loaded onto 10% SDS-PAGE gels.E. coli Rosetta strain . Proteins were induced with 1 mM IPTG (Sigma-Aldrich) for 3 hr at 30°C. Subsequently, proteins were extracted in an appropriate GST lysis buffer  adjusted to 0.1% Triton X-100 and purified to homogeneity with glutathione-agarose beads for 2 hr at 4°C. The beads were then incubated overnight at 4°C and washed with GST lysis buffer adjusted with 1 mM DTT. Subsequently, elution with 10 mM reduced glutathione took place for 15 min at room temperature. Elution was stopped by adding Laemmli buffer and samples were loaded onto the gel after 5 min at 95°C and WB was performed using anti-GST (Sigma no. 06–332) and anti-HA antibodies (Roche no. 11867423001) for immunoblotting.GST-fused recombinant Per2 proteins were expressed in the www.origene.com). The knock-out cell line was produced according to the manufacturer’s protocol. Briefly, cells at 80% of confluency were co-transfected with a donor vector containing the homologous arms and functional cassette, and the guide vector containing the sequence that targets the Cdk5 gene. In parallel, a scrambled negative guide was also co-transfected with a donor vector. 48 hr after transfection the cells were split 1:10 and grown for 3 days. Cells were split another seven times . Then, single colonies were produced and clones were analyzed by PCR in order to find those clones that did not express Cdk5 RNA. Positive clones were re-amplified.The CRISPR/Cas9 Cdk5 cell line was produced starting from NIH3T3 cells using a kit provided by Origene (PCR primers for genomic Cdk5:tgtgagtaccacctcctctgcaa-3’FW: 5’-ttaaacaggccaggcccc-3’RW: 5’-Cdk5 according to the manufacturer’s instructions. The knock-down efficiency was assessed at 48 hr post transduction by western blotting. Scrambled shRNA plasmid (Origene TR30021) was used as a negative control.About 24 hr after seeding cells, different shRNA Cdk5 plasmids (Origene TL515615 A/B/C/D Cdk5 shRNA) were transfected to knock down NIH3T3 cells were treated with 100 µM cycloheximide 48 hr after transfection with the indicated vectors, and cells were harvested 0, 3, and 6 hr after treatment.Cdk5 was silenced were treated for 12 hr with either DMSO (vehicle) or epoxomicin (Sigma-Aldrich) at a final concentration of 0.2 µM. Samples were collected, and proteins extracted followed by western blotting.About 48 hr after transfection with either scrambled or shCdk5, cells where E. coli according to the manufacturer’s protocol described before, using glutathione-sepharose affinity chromatography . Each purified protein (1 µg) was incubated in the presence or absence of recombinant Cdk5/p35 . Reactions were carried out in a reaction buffer  containing [γ-32P] ATP (10 Ci) at room temperature for 1 hr and then terminated by adding SDS sample buffer and boiling for 10 min. Samples were subjected to SDS-PAGE, stained by Coomassie Brilliant Blue, and dried, and then phosphorylated proteins were detected by autoradiography.Recombinant GST-fused PER2 protein fragments were expressed and purified from the BL21 Rosetta strain of 2, and 1 mM DTT) containing [γ-32P] ATP (10 Ci) at room temperature for 1 hr and then terminated by adding SDS sample buffer and boiling for 5 min. Samples were subjected to 15% SDS-PAGE, stained by Coomassie Brilliant Blue, and dried, and then phosphorylated histone H1 was detected by autoradiography. The other half of the IP was used for Western blotting to determine the total amount of CDK5 immunoprecipitated from the SCN samples. To quantify the kinase activity at each time point, the following formula was used: /CDK5 IP protein.CDK5 was immunoprecipitated from SCN samples at different ZTs (circa 600 µg of protein extract) . After i2, and 1.8 mM ATP at 30°C for 1 hr. Samples without ATP were used as negative control. Assays were quenched by 8 M urea and 1 mM DTT. Protein digestion for MS analysis was performed overnight  using a gradient of A (0.1% formic acid in water) and B (0.1% formic acid in 80% acetonitrile in water): loading of sample with 0% B with a flow rate of 600 nL min-1; separation ramp from 5–30% B within 85 min with a flow rate of 250 nL min-1. NanoESI spray voltage was set to 2.3 kV and ion-transfer tube temperature to 250°C; no sheath and auxiliary gas was used. Mass spectrometers were operated in the data-dependent mode; after each MS scan  a maximum of 10 MS/MS scans were performed using a normalized collision energy of 25%, a target value of 1000 and a resolution of 17,500. The MS raw files were analyzed using MaxQuant Software version 1.4.1.2  for peakWe raised in mouse a specific monoclonal antibody recognizing the phosphorylated form of serine 394 of PER2 in collaboration with GenScript Company. The sequence used for the immunogen preparation was: FDY {pSer} PIRFRTRNGEC. 3 Balb/c mice and 3 C57 mice were immunized with conventional strategies and antisera obtained from those animals were used for the first control experiment performed by in vitro kinase assay . The posStatistical analysis of all experiments was performed using GraphPad Prism6 software. Depending on the type of data, either an unpaired t-test, or one- or two-way ANOVA with Bonferroni or Tukey’s post-hoc test was performed. Values considered significantly different are highlighted. . In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:It is clear that post-translational modifications of PER2 are critically important to regulating circadian timing. There are a growing number of modifying enzymes and different type of PTM that form a complex network of regulation. The addition of CDK5 to this network is an important one.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Catherine Dulac as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Cyclin dependent kinase 5 (CDK5) regulates the circadian clock" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Brenna and colleagues provide in this article interesting evidences of the role of CDK5 in the regulation of the circadian clock. Using genetic screen in yeast, viruses-injected animals and knockout and knockdown cells, they clearly show CDK5 shows a rhythmic activity in the SCN, interact with PER2 in a rhythmic fashion, phosphorylates PER2 at serine 394 and regulates its stability and nuclear transport. Finally, they show that Cdk5 knockdown in the SCN of injected animals reduces their circadian period. While authors convincingly show that CDK5 regulates PER2 stability and transport through phosphorylation, the impact of this regulation on the reduced period observed in Cdk5 knockdown animals is less clear. This is confirmed by the fact that this knockdown has the same effect on the period of Per2 mutant animals, suggesting alternative targets that need more attention.However, the contribution of the described pathway to the observed changes of circadian period length upon knockdown of CDK5 in the SCN is not entirely convincing and is based on circumstantial evidence. Specifically, CDK5 could act on CK1δ, as described by the Virshup lab. CK1α which is not phosphorylated at T347 is more active, resulting in a short period phenotype, just as the one observed by the authors.Essential revisions:Brdm1Per2 animals still shows comparable period reduction as knockdown of CDK5 in wild type mice. Brdm1Per2 still express a truncated form of Per2, hence the authors should also consider a similar experiment in Per2 KO animals to prove Per2-dependent mechanism. Alternatively, the CDK5 phenotype in Brdm1Per2animals could be due to CDK5 phosphorylating Per1, which is not discussed by the authors.- Figure 1. The short period phenotype in CDK5 knockdown animals appears to be Per2-independent since knockdown of CDK5 in - Figure 1. Eng, Edison and Virshup, 2017, demonstrated that CKIδ activity towards Per2 is regulated via phosphorylation of T347 by proline-directed kinases . This possibility should be addressed by the authors.- Figure 6. To prove the relevance of this mechanism to other peripheral oscillators, the authors should include an experiment in NIH-3T3 Bmal1-luc cells transfected with shRNA against CDK5.- Mutation of PER2 Ser 394 impacts circadian oscillation.- CDK5 potentially also phosphorylates and regulates PER1 stability and transport.- CDK5 regulation of CLOCK  contributes to the observed phenotype: CLOCK stability and transport are impacted by Cdk5 knockout? Is the circadian effect of Cdk5 knockdown also observed in Clock knockout?While the effect of CDK5 on circadian period is clearly showed, the involvement of PER2 in the mechanism is not clear, meaning the model on Figure 7 is mainly speculative at this stage. Essential revisions:Brdm1 animals still shows comparable period reduction as knockdown of CDK5 in wild type mice.- Figure 1. The short period phenotype in CDK5 knockdown animals appears to be Per2-independent since knockdown of CDK5 in Per2Brdm1Per2mutant mice. As the reviewer states correctly, knockdown of Cdk5 in Brdm1Per2 mutant mice leads to even further shortening of period. The reviewer concludes from this that period shortening by knockdown of Cdk5 is therefore Per2 independent.We think the reviewer is referring to Figure 2 and not Figure 1. Figure 2 shows that knock-down of CDK5 causes short period length that is comparable to the period of Brdm1Per2, allows the identification of genes that are in the same complementation group. If Per2 was the sole target of CDK5 and the phosphorylation important for the period length, then one would expect that the combination of knockdown and knockout would have the same effect as the single conditions. This is not what was observed. However, the effect is not simply additive to indicate two different pathways as suggested by the reviewer. In this scenario, the one-hour reduction of Brdm1Per2 and the one-hour reduction of the knockdown would add up to an at least two-hour shortening and not to the about 1.5 hour shortening as observed. Hence, part of the Cdk5 knockdown effect is mediated by Per2, and this is all what we can/want to say.A genetic complementation assay, in our case the knockdown of Cdk5 in Additionally, our immunofluorescence experiments show, that knockdown of Cdk5 in the SCN prevents PER2 to be localized in the nucleus . If the reviewers view would be correct, this would mean that PER2 protein localization whether in the nucleus or not would be irrelevant for the generation of period length. This, however, is not what genetic experiments revealed, namely that PER2 is a negative feedback loop component of the circadian oscillatory mechanism important to generate circadian period  for which its nuclear shuttling is essential.However, the reviewer has a point, because this experiment shows that knockdown of Cdk5 can affect period not exclusively via PER2 and that there are other Cdk5 targets that may become important in absence of PER2 .Brdm1 still express a truncated form of Per2, hence the authors should also consider a similar experiment in Per2 KO animals to prove Per2-dependent mechanism.Per2Brdm1Per2 mice express a truncated form of PER2. This truncated form of PER2, however, is not in the nucleus, very unstable and degraded fast. We could not detect any PER2 protein in the SCN of Brdm1Per2 mice  similar to our findings in Per2 total KO SCN . This is the reason why the total Per2 ko and the Brdm1Per2 mutant show the same free-running period . Hence, a knockdown of Cdk5 in Per2 KO mice will show the same effect on period as the knockdown in Brdm1Per2 animals.Theoretically, it is correct that Brdm1 animals could be due to CDK5 phosphorylating Per1, which is not discussed by the authors.Alternatively, the CDK5 phenotype in Per2YSPIR in Per2 and PFDHSPIR in PER1, it is likely that CDK5 may also phosphorylate PER1.Since the PER1 and PER2 proteins only differ in a conservative amino acid change one amino acid before the PER2 Ser 394, i. e. PFDHowever, if PER1 would be phosphorylated at the mentioned site above at the same rate as PER2, then also PER1 in addition to PER2 would be absent in the nucleus. This would correspond to a PER1/PER2 double ko. PER1/PER2 ko mice become immediately arrhythmic when they are in constant darkness. This is not the phenotype we observe in the CDK5 knock-down mice and hence it is unlikely that Cdk5 has the same effect on PER1 as it has on PER2. The affinity and efficiency of CDK5 to phosphorylate Per2 may be much higher than that for Per1. However, if PER2 protein is absent (as in the Per2 Brdm mutant) PER1 may become phosphorylated by CDK5 to a greater extent (no competition with PER2) and affect period. Therefore, it will be of great interest to study the relationship between Cdk5, Per1 and Per2. We have discussed this aspect now in the manuscript.- Figure 1. Eng, Edison and Virshup, 2017, demonstrated that CKIδ activity towards Per2 is regulated via phosphorylation of T347 by proline-directed kinases . This possibility should be addressed by the authors.We apologize that this study has escaped our attention. Although we mention that CDK5 may phosphorylate CKI  this study also indicates this. However, both studies do not provide any evidence for CDK5 phosphorylating CKI at T347 in vivo. We cite now both articles in the revised version and depict this possibility in Figure 7.- Figure 6. To prove the relevance of this mechanism to other peripheral oscillators, the authors should include an experiment in NIH-3T3 Bmal1-luc cells transfected with shRNA against CDK5.This is a very good suggestion and we performed the proposed experiment in NIH-3T3 Bmal1-luc cells see  previous- Mutation of PER2 Ser 394 impacts circadian oscillation.This is a very good suggestion, which we also wanted to do. However, overexpressing a PER Ser 394 mutant protein in cell cultures will simply interfere with the circadian clock mechanism in cells, because there is no system at hand that would allow us to express even the normal PER2 protein in a dynamic (circadian) manner. In other words, there is no cell culture system available that would rescue Per2 ko cell lines by overexpression of PER2, since the dynamic presence over time of PER2 is essential for establishing a circadian rhythm. Hence having no rescue system for wild type PER2, it makes no sense to overexpress Ser 394 mutant PER2. Its overexpression simply will interfere with the dynamic time dependent protein-protein interactions of the clock machinery and block it in a similar manner as wild type PER2 would .Brdm1Per2 mutant since the Ser 394 is missing there as well and the PER protein in the Brdm1Per2 mice is very unstable and degraded quickly. Furthermore, a PER2 Ser 394 knock in experiment in mice is for us not feasible to do within 2 months.In vivo a knock-in experiment would be interesting to replace normal PER2 with a Ser 394 PER2 mutant protein. However, we would expect as similar phenotype as in the - CDK5 potentially also phosphorylates and regulates PER1 stability and transport.YSPIR in Per2 and PFDHSPIR in PER1, it is likely that CDK5 may also phosphorylate PER1.This is a highly interesting question . Since the PER1 and PER2 proteins only differ in a conservative amino acid change one amino acid before the PER2 Ser 394, i.e. PFDHowever, if PER1 would be phosphorylated at the mentioned site above and its function would be the same as in PER2, then also PER1 in addition to PER2 would lack in the nucleus. This would correspond to a PER1/PER2 double ko. PER1/PER2 ko mice become immediately arrhythmic when they are in constant darkness. This is not the phenotype we observe in the CDK5 knock-down mice and hence it is unlikely that Cdk5, has the same functional effect on PER1 as it has on PER2. We agree that it remains to be seen what the function of a potential phosphorylation of PER1 by CDK5 might be. This is, however, out of the scope of this manuscript.- CDK5 regulation of CLOCK  contributes to the observed phenotype: CLOCK stability and transport are impacted by Cdk5 knockout?As the reviewer suggests, a study has indicated that CDK5 may phosphorylate CLOCK in vitro (which is cited in the text). If CDK5 regulation of CLOCK in the SCN would play a major role, we would expect the period in wheel running activity to become longer, similar to CLOCK mutant mice . What we observe, however, is a shorter circadian activity period upon knockdown of Cdk5. Therefore, it is unlikely that Clock plays a major role in the behavioral phenotype we observe. Whether NPAS2 can be phosphorylated by CDK5 is unknown and we cannot exclude such a possibility. Interestingly, NPAS2 does not show any consensus sequence that would be recognized by CDK5. Therefore, it is unlikely that CDK5 phosphorylates NPAS2. However, further investigation of the role of CDK5 in the phosphorylation of CLOCK may reveal additional and novel aspects in circadian clock regulation, but is out of the scope of this study.Is the circadian effect of Cdk5 knockdown also observed in Clock knockout?This is related to the question above and very interesting. We have not done an experiment in Clock knock-out mice with a knockdown of Cdk5. I would expect that the longer period phenotype of Clock KO mice would be shortened and appear normal like in wild-type. This is of course a speculation and it remains to be seen what happens in Clock KO mice. Knockdown of Cdk5 would also be interesting to perform in Npas2 KO, Cry KO, Per1 KO, Rev-erba KO and Bmal1 KO mouse strains. However, this is out of the scope of this study.While the effect of CDK5 on circadian period is clearly showed, the involvement of PER2 in the mechanism is not clear, meaning the model on Figure 7 is mainly speculative at this stage.From the questions and answers above it is clear that the regulation of the circadian clock mechanism by CDK5 is more complicated than depicted in the model. We acknowledge this in the revised Figure 7 highlighting the potential additional players that may be influenced by CDK5. However, some aspects in the model concerning PER2 are supported by our findings as well as by previous studies that are cited. A model is always based on what we know. Because we do not know everything of a process the model has to be continuously adapted according to new findings.