IFT57 stabilizes the assembled intraflagellar transport complex and mediates transport of motility-related flagellar cargo

Intraflagellar Transport (IFT) is essential for flagella/cilia assembly and maintenance. Recent biochemical studies have shown that IFT-B is comprised of two subcomplexes, IFT-B1 and IFT-B2. The IFT-B2 subunit IFT57 lies at the interface between IFT-B1 and IFT-B2. Here, using a Chlamydomonas mutant for IFT57, we tested whether IFT57 is critical for IFT-B complex assembly by bridging IFT-B1 and IFT-B2 together. In the ift57-1 mutant, IFT57 and other IFT-B proteins were greatly reduced at the whole-cell level. Strikingly, in the protease free flagellar compartment, while the level of IFT57 was reduced, other IFT particle proteins were not concomitantly reduced but present at the wild-type level. The IFT movement of the IFT57-deficient-IFT particles was also unchanged. Moreover, IFT57 depletion disrupted the flagellar waveform, leading to cell swimming defects. Analysis of the mutant flagellar protein composition showed that certain axonemal proteins were altered. Taken together, these findings suggest that IFT57 does not play an essential structural role in the IFT particle complex but rather functions to prevent it from degradation. Additionally, IFT57 is involved in transporting specific motility-related proteins.</jats:p

Circadian Clock Regulation of the Activity of Translation Initiation Factor eIF2⍺ in Neurospora crassa

At least half of proteins cycling in abundance under control of the circadian clock in eukaryotic cells are synthesized from non-cycling mRNAs. These data suggested that the clock controls posttranscriptional events, including mRNA translation; however, the mechanisms underlying this regulation were not known. We, and other labs, discovered that the circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In Neurospora crassa, the peak in inhibitory eIF2α phosphorylation (P-eIF2α) levels occurs during the subjective day. The activity of the N. crassa eIF2α kinase, CPC-3, was shown to be necessary for rhythmic P-eIF2α accumulation. However, it was not known if CPC-3 activity is sufficient for rhythmic P-eIF2α, or if other factors are involved. In this study, I tested the hypothesis that rhythmic eIF2⍺ activity also requires dephosphorylation of P-eIF2⍺ at night by phosphatases. In support of this hypothesis, I demonstrated that mutation of N. crassa PPP1, a homolog of the yeast eIF2⍺ phosphatase GLC7, leads to high and arrhythmic P-eIF2⍺ levels, while maintaining core circadian oscillator function. Furthermore, PPP1 levels are clock-controlled, peaking at dusk, and rhythmic PPP1 levels were shown to be necessary for rhythmic P-eIF2⍺ accumulation. I discovered that eIF2γ, a component of the eIF2 complex, functions to recruit PPP1 to dephosphorylate eIF2⍺. Thus, in addition to the activity of CPC-3 kinase, rhythmic P-eIF2α requires dephosphorylation by PPP1 phosphatase at night. To determine the impact of rhythmic eIF2α activity on mRNA translation, ribosome profiling, in parallel with RNA-seq, was carried out. In collaboration with Dr. Kathrina Castillo, I found that the N. crassa clock regulates the translation of 1328 mRNAs, of which 404 were translated arrhythmically in strains that lacked CPC-3 (∆cpc-3) or had a constitutively active CPC-3 (cpc-3c). These data revealed that rhythmic eIF2α activity regulates translation of specific, rather than all, mRNAs. Experiments are in progress to investigate the mechanisms of rhythmic P-eIF2α regulation of these targeted mRNAs. Together, these data show how the circadian clock regulates protein production by controlling the activity of a central regulator of translation through the temporal coordination of phosphorylation and dephosphorylation events

The INO80 complex is required for the suppression of WC-independent <i>frq</i> transcription.

<p>(A) ChIP analysis showing WC-2 enrichment at the C-box in the wild-type, <i>ino80</i>^<i>KO</i>, <i>iec-1</i>^<i>KO</i>, and <i>wc-2</i>^<i>KO</i> (<i>bd</i>) strains. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (B) Western blot analysis showing the phosphorylation of WC-1 in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>ino80</i>^<i>KO</i> and <i>iec-1</i>^<i>KO</i> strains. The numbers indicate the ratio of acrylamide/bisacrylamide used in the SDS-PAGE gel. The strains were grown in 2% glucose liquid media. (C) Western blot analysis showing the levels of WC-1 in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>ino80</i>^<i>KO</i> and <i>ies-1</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. (D) Western blot analysis showing the phosphorylation of WC-2 in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>ino80</i>^<i>KO</i> and <i>ies-1</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. (E) Western blot analysis showing the levels of WC-2 in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>ino80</i>^<i>KO</i> and <i>ies-1</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. (F) Western blot analysis of FRQ or WC-1 in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>wc-1</i>^<i>RIP</i> (<i>bd</i>), and <i>ies-1</i>^<i>KO</i> <i>wc-1</i>^<i>RIP</i> strains. The strains were grown in 2% glucose liquid media. (G) Northern blot analysis showing the levels of <i>frq</i> mRNA in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>wc-1</i>^<i>RIP</i> (<i>bd</i>), and <i>ies-1</i>^<i>KO</i> <i>wc-1</i>^<i>RIP</i> strains. The strains were grown in 2% glucose liquid media.</p

IEC-1 suppresses <i>frq</i> transcription and rhythmically binds to the <i>frq</i> promoter.

<p>(A) Western blot analysis showing the circadian oscillation of FRQ proteins in the wild-type and <i>iec-1</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. The asterisk indicates a nonspecific cross-reacted protein band recognized by our FRQ antiserum. The Coomassie Brilliant Blue-stained membranes (mem) represent the total protein in each sample and were used as a loading control. (B) Northern blot analysis of <i>frq</i> transcription in the wild-type and <i>iec-1</i>^<i>KO</i> strains. rRNA was used as a loading control. The strains were grown in 2% glucose liquid media. (C) Immunodetection of IEC-1 protein in the wild-type strain and the <i>iec-1</i>^<i>KO</i> mutant using antiserum that specifically recognizes the IEC-1 protein in the wild-type strain. The arrow notes the specific IEC-1 protein band detected by our IEC-1 antibody. The strains were grown in 2% glucose liquid media. (D) ChIP analysis showing the recruitment of IEC-1 at different regions of the <i>frq</i> locus in the wild-type and <i>iec-1</i>^<i>KO</i> strains at DD18. The strains were grown in 2% glucose liquid media. C-box, clock box; PLRE, proximal light-regulated element; TSS, transcription start site; ORF, open reading frame; UTR, untranslated region. (E) ChIP analysis showing the enrichment of IEC-1 at the C-box of the <i>frq</i> promoter in the wild-type and <i>iec-1</i>^<i>KO</i> strains at the indicated time points. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3).</p

IEC-1 is required for normal circadian clock function.

<p>(A) Race tube assays of the wild-type and <i>iec-1</i>^<i>KO</i> strains. (B) Amino acid sequence alignment of the zf-C2H2 domains of IEC-1 from <i>Neurospora crassa</i>, <i>Penicillium brasilianum</i>, <i>Aspergillus fumigates</i> and <i>Nectria haematococca</i>. (C) Race tube assays of the wild-type strain, <i>iec-1</i>^<i>KO</i> strain, and <i>iec-1</i>^<i>KO</i>, qa-Myc-IEC-1 transformants in a race tube with or without QA. Growth media on the race tubes did not consist of glucose. (D) Luciferase reporter assay showing the <i>frq</i> promoter activity in the <i>wt</i>, <i>frq-luc</i> and <i>iec-1</i>^<i>KO</i>, <i>frq-luc</i> strains grown in DD for several days. Raw data were normalized to subtract the baseline calculated by the LumiCycle analysis software.</p

The establishment of nucleosomal barriers at the <i>frq</i> promoter by the INO80 complex prevents RNA pol II initiation.

<p>(A) ChIP analysis showing H3 density at the C-box, TSS or ORF middle regions of the <i>frq</i> locus in the wild-type, <i>ies-1</i>^<i>KO</i>, and <i>ino80</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (B) ChIP analysis showing the recruitment of SET-2 and enrichment of H3K36me3 at the ORF 3’ of <i>frq</i> in the wild-type and <i>ino80</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3).</p

INO80 is rhythmically recruited at the C-box by IEC-1 and WCC-driven <i>frq</i> expression.

<p>(A) Immunodetection of INO80 in the wild-type strain and the <i>ino80</i>^<i>KO</i> mutant using antiserum that specifically recognizes INO80 protein in the wild-type strain. The strains were grown in 2% glucose liquid media. The arrow indicates the INO80 specific band in the wild-type strains. (B) ChIP analysis showing the recruitment of INO80 at different regions of the <i>frq</i> locus in the wild-type and <i>ino80</i>^<i>KO</i> strains at DD18. The <i>ino80</i>^<i>KO</i> strain was used as a negative control in the ChIP assay. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (C) ChIP analysis showing the recruitment of INO80 at the C-box in the wild-type, <i>ino80</i>^<i>KO</i> and <i>iec-1</i>^<i>KO</i> strains at the indicated time points. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (D) ChIP analysis showing the enrichment of histone H3 at the C-box, PLRE or TSS of the <i>frq</i> promoter region in the wild-type, <i>wc-2</i>^<i>KO</i> (<i>bd</i>) and <i>frq</i>^<i>9</i> (<i>bd</i>) mutant strains. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (E) ChIP analysis showing the recruitment of INO80 at the C-box of the <i>frq</i> promoter in the wild-type, <i>ino80</i>^<i>KO</i>, <i>wc-2</i>^<i>KO</i>(<i>bd</i>), <i>frq</i>^<i>9</i>(<i>bd</i>), and <i>wc-2</i>^<i>KO</i> <i>frq</i>^<i>9</i> (<i>bd</i>) strains at the indicated time points. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (F) Western blot analysis showing the INO80 protein levels in the wild-type, <i>ino80</i>^<i>KO</i>, <i>wc-2</i>^<i>K</i>O (<i>bd</i>), <i>frq</i>^<i>9</i> (<i>bd</i>) and <i>wc-2</i>^<i>KO</i> <i>frq</i>^<i>9</i> (<i>bd</i>) strains. The strains were grown in 2% glucose liquid media.</p