The WD40-repeat protein Pwp1p associates in vivo with 25S ribosomal chromatin in a histone H4 tail-dependent manner

The tails of core histones (H2A, H2B, H3 and H4) are critical for the regulation of chromatin dynamics. Each core histone tail is specifically recognized by various tail binding proteins. Here we screened for budding yeast histone H4-tail binding proteins in a protein differential display approach by two-dimensional gel electrophoresis (2DGE). To obtain highly enriched chromatin proteins, we used a Mg(2+)-dependent chromatin oligomerization technique. The Mg(2+)-dependent oligomerized chromatin from H4-tail deleted cells was compared with that from wild-type cells. We used mass spectrometry to identify 22 candidate proteins whose amounts were reduced in the oligomerized chromatin from the H4-tail deleted cells. A Saccharomyces Genome Database search revealed 10 protein complexes, each of which contained more than two candidate proteins. Interestingly, 7 out of the 10 complexes have the potential to associate with the H4-tail. We obtained in vivo evidence, by a chromatin immunoprecipitation assay, that one of the candidate proteins, Pwp1p, associates with the 25S ribosomal DNA (rDNA) chromatin in an H4-tail-dependent manner. We propose that the complex containing Pwp1p regulates the transcription of rDNA. Our results demonstrate that the protein differential display approach by 2DGE, using a histone-tail mutant, is a powerful method to identify histone-tail binding proteins

Role of the RNA-Binding Protein Nrd1 in Stress Granule Formation and Its Implication in the Stress Response in Fission Yeast

We have previously identified the RNA recognition motif (RRM)-type RNA-binding protein Nrd1 as an important regulator of the posttranscriptional expression of myosin in fission yeast. Pmk1 MAPK-dependent phosphorylation negatively regulates the RNA-binding activity of Nrd1. Here, we report the role of Nrd1 in stress-induced RNA granules. Nrd1 can localize to poly(A)-binding protein (Pabp)-positive RNA granules in response to various stress stimuli, including heat shock, arsenite treatment, and oxidative stress. Interestingly, compared with the unphosphorylatable Nrd1, Nrd1DD (phosphorylation-mimic version of Nrd1) translocates more quickly from the cytoplasm to the stress granules in response to various stimuli; this suggests that the phosphorylation of Nrd1 by MAPK enhances its localization to stress-induced cytoplasmic granules. Nrd1 binds to Cpc2 (fission yeast RACK) in a phosphorylation-dependent manner and deletion of Cpc2 affects the formation of Nrd1-positive granules upon arsenite treatment. Moreover, the depletion of Nrd1 leads to a delay in Pabp-positive RNA granule formation, and overexpression of Nrd1 results in an increased size and number of Pabp-positive granules. Interestingly, Nrd1 deletion induced resistance to sustained stresses and enhanced sensitivity to transient stresses. In conclusion, our results indicate that Nrd1 plays a role in stress-induced granule formation, which affects stress resistance in fission yeast

Phosphorylation of Kif26b Promotes Its Polyubiquitination and Subsequent Proteasomal Degradation during Kidney Development

Kif26b, a member of the kinesin superfamily proteins (KIFs), is essential for kidney development. Kif26b expression is restricted to the metanephric mesenchyme, and its transcription is regulated by a zinc finger transcriptional regulator Sall1. However, the mechanism(s) by which Kif26b protein is regulated remain unknown. Here, we demonstrate phosphorylation and subsequent polyubiquitination of Kif26b in the developing kidney. We find that Kif26b interacts with an E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) in developing kidney. Phosphorylation of Kif26b at Thr-1859 and Ser-1962 by the cyclin-dependent kinases (CDKs) enhances the interaction of Kif26b with Nedd4. Nedd4 polyubiquitinates Kif26b and thereby promotes degradation of Kif26b via the ubiquitin-proteasome pathway. Furthermore, Kif26b lacks ATPase activity but does associate with microtubules. Nocodazole treatment not only disrupts the localization of Kif26b to microtubules but also promotes phosphorylation and polyubiquitination of Kif26b. These results suggest that the function of Kif26b is microtubule-based and that Kif26b degradation in the metanephric mesenchyme via the ubiquitin-proteasome pathway may be important for proper kidney development

p54nrb/NonO and PSF promote U snRNA nuclear export by accelerating its export complex assembly.

The assembly of spliceosomal U snRNPs in metazoans requires nuclear export of U snRNA precursors. Four factors, nuclear cap-binding complex (CBC), phosphorylated adaptor for RNA export (PHAX), the export receptor CRM1 and RanGTP, gather at the m(7)G-cap-proximal region and form the U snRNA export complex. Here we show that the multifunctional RNA-binding proteins p54nrb/NonO and PSF are U snRNA export stimulatory factors. These proteins, likely as a heterodimer, accelerate the recruitment of PHAX, and subsequently CRM1 and Ran onto the RNA substrates in vitro, which mediates efficient U snRNA export in vivo. Our results reveal a new layer of regulation for U snRNA export and, hence, spliceosomal U snRNP biogenesis

N-Terminal Phosphorylation of HP1α Promotes Its Chromatin Binding ▿

The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1α plays a central role in its targeting to chromatin. Recombinant HP1α prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1α was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1α's affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1α to H3K9me by mediating the interaction between HP1α and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1α exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1α's N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications

MOESM2 of RNAi-dependent heterochromatin assembly in fission yeast Schizosaccharomyces pombe requires heat-shock molecular chaperones Hsp90 and Mas5

Additional file 2: Table S1. Strains used in this study. Table S2. Primers used in this study. Table S3. Transcription related factors detected in massspectrometry analysis. Table S4. List of candidate genes for silencing factors suggested by massspectrometry

MOESM1 of RNAi-dependent heterochromatin assembly in fission yeast Schizosaccharomyces pombe requires heat-shock molecular chaperones Hsp90 and Mas5

Additional file 1: Figure S1. Fission yeast Hsp70 proteins and their homologs. (A) Phylogenic tree of Hsp70 proteins. Scale-bar unit indicates the number of amino acid substitutions per site. Names of proteins are associated with two-letter abbreviations and color-coded to indicate the species: “sp” for Schizosaccharomyces pombe (red), “sc” for Saccharomyces cerevisiae (black), and “dm” for Drosophila melanogaster (blue). (B) Domain structure of Hsp70 proteins. Protein names are depicted as in (A) and their amino acid lengths are shown. Protein domains are drawn as boxes according to the CATH-Gene3D classification. The N-terminal ATPase domains (ATPase) are composed of three internal domains that belong to the CATH superfamilies 3.30.420.40, 3.30.30.30, and 3.90.640.10. Substrate-binding domains (SB) belong to the 2.60.34.10 superfamily. The C-terminal lid domains (Lid) belong to the 1.20.1270.10 superfamily. Predicted localization signals for mitochondria (TargetP-M) and for the endoplasmic reticulum or beyond (TargetP-S) are shown as shaded boxes. Locations of the proteins in the cell are indicated. For clarity, D. melanogaster Hsp70 proteins other than Hsc70-4 are omitted. Figure S2. Hsp40 family proteins in fission yeast. Domain structure of all 26 fission yeast Hsp40 proteins are shown. Protein names and amino acid lengths are indicated. Protein domains are drawn as boxes according to the CATH-Gene3D, Pfam, and Prosite classifications. Predicted localization signals are shown as in Additional file 1: Figure S1. Predicted trans-membrane helix regions (TMhelix) are indicated as gray boxes. The DnaJ domains belong to the CATH superfamily 1.10.287.110. Type-I Hsp40 proteins are characterized by C-terminal (purple) and central (pink) domains that belong to the CATH superfamilies 2.60.260.20 and 2.10.230.10, respectively. The type-II Hsp40 protein Psi1 also contains the C-terminal domain but lacks the central domain. The other proteins are classified as the type-III Hsp40 proteins. Figure S3. Fission yeast type-I and -II Hsp40 proteins and their homologs. (A) Phylogenic tree of Hsp40 proteins. Scale-bar unit indicates the number of amino acid substitutions per site. Names of proteins are depicted as in Additional file 1: Figure S1. (B) Domain structure of Hsp40 proteins. Protein names are depicted as in (A) and amino acid lengths are shown. Protein domains and predicted localization signals are drawn as in Additional file 1: Figure S2. Locations of proteins in the cell are indicated. For clarity, D. melanogaster Hsp40 proteins other than Droj2 are omitted. Figure S4. Schematic representation of marker integration sites. The ade6+ and ura4+ marker genes were located in the SphI site of otr1R (otr1R(SphI)::ade6+) and in the NcoI site of imr1L (imr1L(NcoI)::ura4+), respectively. Figure S5. Hsp90 and Mas5 are dispensable for red pigment formation. Cells were spotted on normal YES plates (YES) and YES containing limited amount of adenine (Low adenine), and incubated at 30°C for six days. Figure S6. Derepression of marker genes inserted in the pericentromere. (A) Cells were serially diluted, spotted on normal EMMS plates and EMMS lacking adenine (EMMS - adenine) or uracil (EMMS - uracil), and incubated at 30°C for 6 days. (B) Strand-specific RT-qPCR for the ura4 marker gene. Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (n = 3). (C) Strand-specific RT-qPCR for the ade6 and ura4 genes located in the endogenous loci (ade6+ and ura4+) and in the pericentromere (otr1R::ade6+ and imr1L::ura4+). Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (n = 3). **P < 0.01 (Student’s t-test). Figure S7. Hsp90 and Mas5 are dispensable for the silencing at the mating-type locus. Strand-specific RT-qPCR for the cenH transcript from the mating-type locus. Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (n = 6). **P < 0.01 (Student’s t-test). Figure S8. Reduction of Ago1 protein level in hsp90-A4 and mas5∆ cells. Two-fold serially diluted whole-cell extracts were separated by SDS-PAGE followed by western blotting and Coomassie brilliant blue staining. The membrane for detecting FLAG-Ago1 with an antibody against FLAG epitope was reprobed with an antibody against α-tubulin. Figure S9. mRNA expression levels of arb1 and tas3 genes. Strand-specific RT-qPCR for the arb1 and tas3 genes. Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (n = 6). *P < 0.05, **P < 0.01 (Student’s t-test). Figure S10. Protein expression level of Tas3. Detection of proteins in whole-cell extracts by western blotting. The membrane for detecting Tas3-Myc with an antibody against Myc epitope was reprobed with an antibody against α-tubulin to confirm that equal amounts of samples were loaded. For the mas5 deletion, four independently constructed clones were tested. Figure S11. Colony colors of otr1R::ade6+ strains are dependent on the ade6 alleles in the endogenous ade6 locus. Cells were streaked on normal YES plates (YES) and YES containing limited amount of adenine (Low adenine), and incubated at 30°C for four days. Strains with otr1R::ade6+ in the ade6-DN/N genetic background formed darker colonies. Note that the colony colors of canonical heterochromatin mutants (i.e. clr4∆) in the ade6-m210 background were faint pink, and are not as white as those of strains with the native ade6+ allele in the endogenous ade6 locus. Figure S12. Reintroduction of the R33C mutation phenocopied the original A4 isolate. (A) Schematic diagram showing the introduction of the mutation (R33C) found in the original A4 isolate. (B) Cells were streaked on normal YES plates (YES) and YES containing limited amount of adenine (Low adenine), and incubated at 30°C for four days. In HKM-1565 and HKM-1618, the G418-resistant kanMX cassette was located upstream of the hsp90 promoter. Both strains were generated by introducing kanMX-containing DNA fragments with or without the R33C mutation into the genome of the wild-type strain HKM-1100. Figure S13. Silver staining of immunoprecipitated proteins that were analyzed by mass spectrometry. Immunoprecipitates from indicated cells were separated by SDS-PAGE and silver-stained (see Methods). Each lane in the gel was sliced as indicated by red lines. Proteins in each gel piece were analyzed by nano-liquid chromatography tandem mass spectrometry. Black arrows indicate visible protein bands that are absent or barely seen in the untagged control (no-tag)

CDKs phosphorylate Thr-1859 and Ser-1962 on Kif26b.

<p><i>A.</i> Schematic diagram of the amino acids in Kif26b potentially phosphorylated by CDK5. The C-terminal tail region of Kif26b that interacts with Nedd4 is indicated by the bar. Underlined amino acids indicate CDK5 phosphorylation sites. <i>B.</i> GST or GST-Kif26b-C was incubated with or without recombinant His-tagged CDK1, CDK2, CDK5 or CDK6 and a kinase assay was performed. Proteins were separated by SDS-PAGE and detected by Coomassie Brilliant Blue (CBB) staining or immunoblotting with the indicated antibodies. <i>C</i>. HEK293 cells were transfected with a FLAG-Kif26b-expressing plasmid. At 48 hrs post-transfection, the cells were treated for 6 h with DMSO (-) or Roscovitine (+; 20 µM). FLAG-tagged proteins were immunoprecipitated from the cell lysates with anti-FLAG beads. Precipitates were then treated with or without calf intestine-derived alkaline phosphatase (AP) at 37°C for 1 h and analyzed by immunoblotting with the indicated antibodies. <i>D</i>. HEK293 cells were transfected with WT or substitution mutant FLAG-Kif26b-expressing plasmids. The FLAG-tagged proteins were immunoprecipitated from the cell lysates with anti-FLAG beads. The precipitates were analyzed by immunoblotting with the indicated antibodies.</p

Phosphorylation and polyubiquitination of Kif26b are induced by disruption of microtubules.

<p><i>A.</i> HeLa cells stably expressing FLAG-Kif26b were pretreated for 3 h with DMSO or Roscovitine (20 µM) and then treated for the indicated time with 5 µM nocodazole (left panel). HEK293 cells stably expressing FLAG-Kif26b were pretreated for 3 h with DMSO or Roscovitine (20 µM) and then treated for 6 h with 5 µM nocodazole (right panel). The lysates were subjected to immunoblotting with the indicated antibodies. <i>B.</i> HeLa cells stably expressing FLAG-Kif26b were treated for 6 h with nocodazole (5 µM), and then the FLAG-tagged proteins were immunoprecipitated from the cell lysates with anti-FLAG beads. The precipitates were subsequently treated with or without AP at 37°C for 1 h and analyzed by immunoblotting with anti-FLAG antibody. <i>C</i>. HeLa or HEK293 cells stably expressing FLAG-Kif26b (left and right panel, respectively) were treated as in <i>B,</i> and then the FLAG-tagged proteins were immunoprecipitated from the cell lysates with anti-FLAG beads. The precipitates were analyzed by immunoblotting with the indicated antibodies. <i>D</i>. HeLa or HEK293 cells stably expressing FLAG-Kif26b (left and right panel, respectively) were pretreated for 6 h with nocodazole and then further treated for 8 h with MG132 (20 µM). The FLAG-tagged proteins were immunoprecipitated from the lysates with anti-FLAG beads and subjected to immunoblotting with the indicated antibodies. <i>E</i>. HeLa or HEK293 cells stably expressing FLAG-Kif26b (left and right panel, respectively) were treated for 8 h with DMSO, MG132 (20 µM), or chloroquine (100 µM). Whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies.</p

Kif26b is an unconventional kinesin.

<p><i>A.</i> Alignment of the amino acid sequences of the motor domains of various KIFs. Human KIF5A, GAKIN/KIF13B, KIF26A, and KIF26B and mouse Kif26b are shown. Amino acids that correspond to the p-loop, L9-loop, and L11-loop consensus sequences are shown on a gray background. <i>B.</i> Schematic diagram of the microtubule-binding assay procedure (left panel). COS-7 cells were transfected with FLAG-Kif26b- or FLAG-GAKIN/KIF13B-expressing plasmids. At 48 h after post-transfection, the microtubule-binding assay was performed on the cell lysates and the precipitates were analyzed by immunoblotting with anti-FLAG antibody (right panel). <i>C.</i> HeLa cells stably expressing FLAG-Kif26b were treated for 1 h with DMSO or nocodazole (5 µM). The cells were then fixed and stained with anti-FLAG (green) and anti-β-tubulin (red) antibodies. Scale bar = 10 µm.</p