Circadian clock control of tRNA synthetases in Neurospora crassa

Background: In Neurospora crassa, the circadian clock controls rhythmic mRNA translation initiation through regulation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2). Active CPC-3 phosphorylates and inactivates eIF2α, leading to higher phosphorylated eIF2α (P-eIF2α) levels and reduced translation initiation during the subjective day. This daytime activation of CPC-3 is driven by its binding to uncharged tRNA, and uncharged tRNA levels peak during the day under control of the circadian clock. The daily rhythm in uncharged tRNA levels could arise from rhythmic amino acid levels or aminoacyl-tRNA synthetase (aaRSs) levels. Methods: To determine if and how the clock potentially controls rhythms in aspartyl-tRNA synthetase (AspRS) and glutaminyl-tRNA synthetase (GlnRS), both observed to be rhythmic in circadian genomic datasets, transcriptional and translational fusions to luciferase were generated. These luciferase reporter fusions were examined in wild type (WT), clock mutant Δ frq, and clock-controlled transcription factor deletion strains. Results: Translational and transcriptional fusions of AspRS and GlnRS to luciferase confirmed that their protein levels are clock-controlled with peak levels at night. Moreover, clock-controlled transcription factors NCU00275 and ADV-1 drive robust rhythmic protein expression of AspRS and GlnRS, respectively. Conclusions: These data support a model whereby coordinate clock control of select aaRSs drives rhythms in uncharged tRNAs, leading to rhythmic CPC-3 activation, and rhythms in translation of specific mRNAs.


Introduction
Aminoacyl-tRNA synthetases (aaRSs) play a fundamental role in mRNA translation by catalyzing the attachment of specific amino acids onto their cognate tRNAs. For accuracy, aaRSs employ chemical selectivity and proofreading capabilities (Rubio Gomez andIbba, 2020, Roux andTopisirovic, 2012). Mounting evidence supports that aaRSs have functions beyond their role in charging tRNAs, including roles in immune signaling, cell cycle, nutrient metabolism and growth, and thus are linked to various human diseases (Pang et al., 2014, Nie et al., 2019, Park et al., 2008. Aminoacylation mechanisms are conserved across all kingdoms of life. The regulation of aaRS expression in prokaryotes is well-described (Green et al., 2010, Pelchat andLapointe, 1999), and while less is known about their regulation in eukaryotes, coordinate induction of several mammalian cytoplasmic aaRS genes by amino acid starvation has been observed (Shan et al., 2016). In addition, several aaRSs were reported to have daily rhythms in abundance at the mRNA and/or protein levels in the filamentous fungus Neurospora crassa (Sancar et al., 2015, Hurley et al., 2018, Castillo et al., 2022b, and mammalian cells (Pembroke et al., 2015, Barclay et al., 2012, Hughes et al., 2009, Miller et al., 2007, Vollmers et al., 2012, Eckel-Mahan and Sassone-Corsi, 2009, Geyfman et al., 2012, Yoshitane et al., 2014, Janich et al., 2015. These data suggested that the circadian clock imparts regulation on aars gene expression, which would impact rhythmic protein synthesis and clock-controlled cellular processes.
The circadian clock is an endogenous timekeeping mechanism that regulates diverse biological processes in many organisms, allowing them to anticipate and prepare for daily environmental cycles, and to organize cellular processes to the right time of day for improved fitness (Dunlap and Loros, 2017). Disruption of the circadian clock has profound effects on human physiology and behavior, and can lead to a wide range of diseases (Bass, 2017, Foster, 2020, Hernandez-Garcia et al., 2020. Depending on the organism and tissue type, the circadian clock regulates daily rhythms in mRNA and protein accumulation for up to 50% of the eukaryotic genome (Hurley et al., 2014, Zhang et al., 2014, Mauvoisin et al., 2014. Remarkably, most of the proteins that cycle in abundance under the control of the circadian clock are produced from mRNAs that are not clock-controlled (Reddy et al., 2006, Robles et al., 2014, Zhang et al., 2014, Hurley et al., 2018, Castillo et al., 2022b. These data suggested a prominent role for clock regulation of posttranscriptional processes, including rhythmic mRNA translation.
The N. crassa circadian clock is composed of negative elements FREQUENCY (FRQ), FRQ-INTERACTING RNA HELICASE (FRH), and CASEIN KINASE 1 (CK1), and positive elements WHITE COLLAR-1 (WC-1) and WHITE COLLAR-2 (WC-2) (Baker et al., 2012, Dunlap andLoros, 2017). WC-1 and WC-2 heterodimerize to form the White Collar Complex (WCC) which binds to the promoters of frq and downstream clock-controlled genes (ccgs), including 24 transcription factors, to drive their rhythmic transcription (Froehlich et al., 2003, Smith et al., 2010. In addition, the N. crassa clock generates rhythms in the activities of the conserved eukaryotic translation initiation factor 2 (eIF2) and eukaryotic translation elongation factor 2 (e-EF2) (Caster et al., 2016, Karki et al., 2020, Ding et al., 2021. A central mechanism for translational control is the phosphorylation of eIF2α, as even partial phosphorylation is sufficient to inhibit protein synthesis (Baird and Wek, 2012). Furthermore, rhythms in activity of the N. crassa eIF2α kinase CPC-3, a homolog of the well-studied yeast and mammalian eIF2α kinase GCN2, are dependent on rhythmic uncharged tRNA Val , levels. The rhythm in uncharged tRNA Val levels is driven, at least in part, by rhythms in valyl-tRNA synthetase (ValRS) levels (Karki et al., 2020). However, in addition to ValRS, several other aaRSs were found to be clock-controlled from genomic datasets (Sancar et al., 2015, Hurley et al., 2018, Castillo et al., 2022b, suggesting that aaRSs may be coordinately regulated by the clock to control rhythmic translation. In this study, we sought to independently validate clock control of two aaRSs and begin to examine the mechanisms of clock control of aaRS genes. Using aspartyl-tRNA synthetase (AspRS) and glutaminyl-tRNA synthetase (GlnRS)

REVISED Amendments from Version 1
In the revised manuscript, ribo-seq heatmaps of all aaRS were added to Figure 1A to show both rhythmic and nonrhythmic ribosome occupancy. New control data showing that both Pasprs::luc and Pglnrs::luc are arrhythmic in clock mutant Δfrq cells were added to Figure 2 (D and E). The strains and additional data were generated and analyzed by Teresa M. Lamb and Madhusree Gangopadhyay, both of whom were added as authors. The data from Figure 4C examining GlnRS rhythms in Δadv-1 cells was reanalyzed to include only the time points from DD12 to DD96. These data fit to a sine wave with a period of 22.5 h, but with a low amplitude compared to WT. This information is now included in Table 2, and we clarify in the text that deletion of transcription factor ADV-1 decreased the amplitude of GlnRS rhythms. In the text we added background information on why particular transcription factors were chosen for this study, added an updated reference on what is known regarding the regulation of eukaryotic aaRS genes, and provided a brief description of published Ribo-seq datasets used in this study in the methods.
Any further responses from the reviewers can be found at the end of the article luciferase translational reporters, we confirmed that AspRS and GlnRS protein levels are rhythmic in WT cells with a peak in the subjective night, similar to the peak time of ValRS (Karki et al., 2020), and arrhythmic in clock mutant Δfrq cells. We identified clock-controlled transcription factors that regulate AspRS::LUC and GlnRS::LUC rhythmic accumulation. We show that AspRS levels are low and arrhythmic in cells deleted for transcription factor NCU00275, and GlnRS levels are high and display a low amplitude rhythm in cells deleted for transcription factor ADV-1. These findings provide a basis for further studies investigating coordinate clock control of aaRSs via different clock-controlled transcription factors and the roles of rhythmic aaRS's in rhythmic mRNA translation.

Luciferase assays
To examine bioluminescence rhythms arising from strains containing luciferase fusions, 5 μl of a 1Â10 5 conidia/ml suspension were inoculated into 96 well microtiter plates containing 150 μl of 1X Vogel's salts, 0.01% glucose, 0.03% arginine, 0.1 M quinic acid, 1.5% agar, and 25 μM firefly luciferin, pH 6. After inoculation of conidia (1Â10 5 conidia), the microtiter plate was incubated at 30°C in LL for 24 h and transferred to DD 25°C to obtain bioluminescence recordings using EnVision Xcite Multilabel Reader (Perkin-Elmer), with recordings taken every 90 min over 4-5 days. Raw reads were normalized to the mean to graph the data.

Sequencing datasets
Rhythmic expression of N. crassa clock-controlled tRNA synthetases was determined using public ribosome profiling (ribo-seq) and RNA-seq datasets for WT and clock mutant Δfrq cells (Castillo et al., 2022b). Briefly, ribo-seq and RNAseq were performed in parallel from cells grown in constant darkness (DD) in a circadian time course with 4-h resolution for two biological replicates.
Statistical test for rhythmicity and analysis of circadian parameters Rhythmic data from luciferase assays were fit to a sine wave or a line as previously described (Lamb et al., 2011). Nonlinear regression to fit the rhythmic data to a sine wave (fitting period, phase, and amplitude) and a line (fitting slope and intercept), as well as Akaike's information criteria tests to compare the fit of each data set to the 2 equations, were carried out using the Prism software package version 9.4.0. The p-values reflect the probability that, for instance, the sine wave fits the data better than a straight line. Error bars in all graphs represent the standard error of the mean (SEM) from independent experiments. Raw and normalized luciferase activity reads were analyzed for period, phase, and amplitude values using BioDare version 2 (Zielinski et al., 2014). Heat maps were generated using the ggplot2 R package for genes with rhythmic ribosome protected fragments (RPF) counts in WT, and sorted according to increasing peak phase of the oscillation (Wickham, 2016). ribosome protected fragments (RPF) levels are standardized within each gene (row) (Z-scores).

AspRS and GlnRS protein levels are clock-controlled
Published N. crassa circadian ribosome profiling (ribo-seq) data revealed rhythms in ribosome occupancy for 17 of 36 aaRS using the Extended Circadian Harmonic Oscillator (ECHO) rhythmicity detection tool (De Los Santos et al., 2020, Castillo et al., 2022b. Genes with an adjusted p-value of < 0.05, and with circadian harmonic, damped, or forced oscillation types were considered rhythmic. A heat map of the phase-sorted fitted ribosome protected footprint (RPF) values obtained using ECHO showed robust rhythmic ribosome occupancy for 17 aaRSs in WT cells, with peak ribosome occupancy primarily during the late subjective day (DD40-44) ( Figure 1A, Class I). As expected for circadian clock control, the rhythms were abolished in the clock mutant Δfrq cells. Class II aaRS genes were arrhythmic in both WT and Δfrq cells ( Figure 1A). Class III aaRS genes were rhythmic in both WT and Δfrq cells, suggesting that the rhythms are controlled by something other than the FRQ/WCC circadian oscillator ( Figure 1A). Circadian rhythms in ValRS protein levels were previously validated using a luciferase (LUC) translational reporter. ValRS::LUC levels peaked in the subjective night (Karki et al., 2020), lagging the observed peak in ribosome occupancy ( Figure 1A).
In higher eukaryotes, 9 aaRSs form a multisynthetase complex (MSC) that is proposed to aid translation by providing a channel through which tRNAs can pass to reach bound aaRSs (Hyeon et al., 2019). Interestingly, 5 of the 9 aaRSs in the complex (AspRS, GlnRS, GluRS, LeuRS, and MetRS) are clock-controlled based on our ribosome profiling datasets ( Figure 1A), and we focused on validating circadian clock control of AspRS and GlnRS ( Figure 1B & C). AspRS and GlnRS luciferase translational reporter fusions were generated (AspRS::LUC and GlnRS::LUC) and assayed for rhythmic luciferase levels from cells grown in DD over 4 days ( Figure 1D & C). Bioluminescence rhythms were observed for both AspRS::LUC and GlnRS::LUC, with peak levels during the early subjective night (e.g. DD 48) and a period of 22.4 AE 0.6 h and 22.0 AE 0.4 h, respectively (Table 2). Similar to ValRS::LUC, the peak in AspRS::LUC and GlnRS::LUC levels occurred a few hours after the peak in ribosome occupancy ( Figure 1B-E). The AspRS::LUC and GlnRS::LUC rhythms were abolished in Δfrq cells, confirming that AspRS and GlnRS protein levels are clock-controlled ( Figure 1D & E, Table 2).

Clock-controlled transcription factors drive rhythms in AspRS and GlnRS expression
In addition to rhythms in protein levels, several N. crassa aaRS mRNAs were reported in genome-wide studies to be clock-controlled (Hurley et al., 2014, Sancar et al., 2015, Castillo et al., 2022b. Of these aaRSs, asprs and glnrs exhibited rhythms in mRNA levels in WT cells, with mRNA levels peaking in the subjective early evening (DD40-44) (Figure 2A & B). In support of these genomic data, asprs (Pasprs::luc) and glnrs (Pglnrs::luc) promoter luc fusions were rhythmic in DD peaking during the subjective night ( Figure 2C & D), with no significant period and phase differences between the mRNA and protein levels ( Table 2).  As expected for clock control, the mRNA rhythms were abolished in Δfrq cells as shown by the ECHO-generated fitted values for normalized mRNA levels by FPKM (fragments per kilobase of exon per million mapped reads) and by aars promoter luciferase reporter fusion assays (Figure 2A & B) (Castillo et al., 2022b). Together, these data support that rhythmic AspRS and GlnRS protein levels arise, at least in part, from clock-controlled mRNA levels.
To determine if clock-controlled rhythms in AspRS and GlnRS protein levels require clock-controlled transcription factors and rhythmic transcription, we examined AspRS::LUC and GlnRS::LUC rhythms in cells deleted for transcription factors that are direct targets of the WCC. The levels of the transcription factor mRNAs peak in the subjective morning and the transcription factors bind to downstream ccgs to regulate their rhythmic expression (Smith et al., 2010, Dekhang et al., 2017, Munoz-Guzman et al., 2021. Three clock-controlled transcription factors were selected for this initial study. CLR-1 (NCU07705) is a zinc binuclear cluster transcription factor that is important in nutrient sensing and signaling, and was shown to be necessary for normal AspRS mRNA levels when N. crassa cells were grown on cellulose (Coradetti et al., 2012). However, AspRS::LUC levels were rhythmic in Δclr-1 cells with a period and phase, and overall levels, that were similar to WT when the cultures were grown in DD ( Figure 3A, Table 2). Transcription factor NCU00275 was selected as a representative clock-controlled transcription factor of unknown function (Smith et al., 2010). AspRS::LUC rhythms were abolished in Δncu00275 cells ( Figure 3B, Table 2) and AspRS::LUC levels were lower in Δncu00275 compared to WT cells ( Figure 3C). These data supported that NCU00275 is necessary for circadian rhythms in AspRS::LUC levels and directly, or indirectly, activates asprs transcription. GlnRS::LUC was rhythmic in Δncu00275 cells with no significant differences in levels, period or phase between WT and Δncu00275 ( Figure 4A, Table 2). Previous RNA-seq data comparing WT to cells deleted for the transcription factor ADV-1 (NCU07392) showed that the levels of glnrs mRNA, but not asprs mRNA levels, were generally higher in Δadv-1 cells compared to WT grown in DD and following light treatments (Dekhang et al., 2017). ADV-1 ChIP-seq data did not reveal significant binding of ADV-1 to the promoter of glnrs, suggesting indirect negative regulation of glnrs by ADV-1. Based on these data, we examined GlnRS::LUC rhythms in Δadv-1, and found that GlnRS::LUC rhythms became progressively damped by day 3 in DD compared to WT ( Figure 4B), leading to a significantly reduced amplitude of oscillation ( Figure 4C, Table 2). Consistent with the RNA-seq data, the raw bioluminescence signals showed that GlnRS::LUC levels were higher in Δadv-1 than in WT cells ( Figure 4D). Taken together, these data support that specific clock-controlled transcription factors contribute to the rhythmic expression of aaRSs.
All underlying data can be found in the Underlying data section (Castillo et al., 2022b, Castillo andBell-Pedersen, 2022).

Discussion
aaRSs play a central role in translation and translation fidelity, yet the regulation of aaRS gene expression in eukaryotes is understudied. Genome-wide datasets revealed that several eukaryotic aaRSs are clock-regulated at the level of mRNA and protein (Sancar et al., 2015, Hurley et al., 2018, Castillo et al., 2022b, Barclay et al., 2012, Pembroke et al., 2015, Hughes et al., 2009, Miller et al., 2007, Vollmers et al., 2012, Eckel-Mahan and Sassone-Corsi, 2009, Geyfman et al., 2012, Yoshitane et al., 2014, Janich et al., 2015. We previously showed that the levels of ValRS cycle under control of the circadian clock with peak levels during the subjective night (Karki et al., 2020). Here, we validated that asprs and glnrs mRNA and protein levels are also clock-controlled with a similar night-time peak in protein levels. The bulk of rhythmic protein accumulation occurs at night in N. crassa (Hurley et al., 2018) supporting that the night-time peak in aaRS protein levels serve to coordinately increase protein synthesis at night.
We observed that AspRS and GlnRS protein rhythms are dependent on the circadian clock through the activities of clockcontrolled transcription factors. AspRS levels are arrhythmic and low in Δncu00275, suggesting that NCU00275 activates asprs transcription. NCU00275 is annotated as a hypothetical protein, but its homologs in other fungi suggest that it is a C3HC4-type RING finger protein involved in transcription, signal transduction, ubiquitination, and recombination (Basenko et al., 2018, Krishna et al., 2003. NCU00275 mRNA levels peak in the subjective morning under control of the WCC (Smith et al., 2010), whereas asprs mRNA and protein levels peak several hours later in the early subjective night. This delay suggests the possibility that the effect of NCU00275 on asprs transcription is indirect, requires other transcription factors or changes in chromatin state, and/or that posttranscriptional regulation of asprs contributes to this delay. Furthermore, deletion of NCU00275 had no significant effect on GlnRS levels, indicating that coordinate regulation of the aaRS's by the clock is not necessarily through the same clock-controlled transcription factors. Consistent with this idea, the clock-controlled transcription factor ADV-1 was previously shown to affect the levels of glnrs, but not asprs, mRNA levels (Dekhang et al., 2017), and GlnRS levels and rhythms were altered in Δadv-1 cells compared to WT. Furthermore, ChIP-seq data showed that ADV-1 binds to the promoter of TrpRS (NCU06722) and MetRS (NCU07451), both of which are clock-controlled, and to the promoter of LysRS (Dekhang et al., 2017), which was not rhythmic in our ribo-seq data (Figure 1). Additional experiments will be done in the future to confirm clock control of the other N. crassa aaRS's predicted to be rhythmic from RNA-seq and ribo-seq datasets, and to comprehensively define the roles of clock-controlled transcription factors in aaRS regulation.
Charging tRNAs with the correct amino acid is the first step in translation, and therefore the levels and function of aaRSs are critical to translation fidelity (Yu et al., 2021, Hausmann andIbba, 2008). Mistakes in translation are generally considered detrimental; however, during stress, mistranslation may be beneficial by increasing the levels of altered proteins that can perform new functions to aid the response (Pan, 2013, Ribas de Pouplana et al., 2014. In eukaryotes, some aaRSs form the MSC (Bandyopadhyay and Deutscher, 1971, Lee et al., 2004, Kerjan et al., 1994, with varying composition dependent on the organism. In S. cerevisiae, the MSC is comprised of MetRS, GluRS and the scaffold protein Arc1 (Galani et al., 2001). The MSC in mammals has 9 aaRSs, including 5 of the 9 aaRSs (AspRS, GlnRS, GluRS, LeuRS, and MetRS) that are rhythmic in ribo-seq data sets, and 3 scaffold proteins, AIMP1-3. The MSC helps the function of its components; for example, the K m for binding of tRNA Met to MetRS in the yeast MSC is about 100-fold lower compared to the K m for binding of tRNA Met to MetRS alone (Simos et al., 1998). MSC components are also involved in cell signaling, stress responses, metabolite sensing, and controlling gene expression by binding to specific RNA and DNA sites, supporting the idea that a key role of the MSC is to support alternative functions of aaRSs (Cui et al., 2021, Pang et al., 2014, Rubio Gomez and Ibba, 2020. Furthermore, aaRSs, either alone or in the MSC complex, participate in a wide variety of processes outside of their classical role in tRNA charging, including transcription regulation, splicing, and metabolism (Rubio Gomez and Ibba, 2020), and abnormal expression, localization, and molecular interactions of aaRSs are associated with a variety of human diseases, including cancer (Zhou et al., 2020). This widespread impact of aaRSs on host biology raises the intriguing idea that daily rhythms in the levels of aaRSs represent a missing factor linking the clock to a wide range of rhythmic biological processes that are critical to health, underscoring the need to better understand the mechanisms underlying their circadian regulation.

Data availability
Underlying data Gene Expression Omnibus: Ribosome profiling and RNA-seq data used in Figures 1 and 2 This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Luis F. Larrondo
Millennium Science Initiative, Millennium Institute for Integrative Biology (iBio), Santiago, Chile This reviewer appreciates the efforts made by the authors to address the prior points.
It is surprising to see that the Class III aaRS show rhythms in Dfrq, which are overall just like WT (not amplitude nor phase appear to be affected).
Hopefully the authors will tackle some of those aaRS in future studies.
Could the authors indicate the p value for Dfrq, as visual inspection of the data shows a ":hint" of rhythmicity and therefore it would be informative to know if p value is 0.06 or 0.6 for example) "We found, for example, that AspRS::LUC was rhythmic in WT and Δclr-1 (NCU07705) cells".
It is unclear why the authors decided to examine these two particular KOs (NCU07705 and NCU00275) in AspRS expression. Was the choice guided by bioinformatic analyses or literature curation? The same applies to the GlnRS.

4.
"Furthermore, the progressive dampening of GlnRS::LUC levels in Δadv-1 significantly reduced the amplitude of oscillation, leading to arrhythmicity ( Figure 4D)." While I agree that the absence of adv-1 has an impact on GlnRS expression, I am not so convinced that the end result is arrhythmic gene expression.
Eye-examination of Fig 4c reveals a distinct oscillatory pattern. I would even predict that if the rhythmicity analysis is limited to the first 72 or 96 h, the software will confirm circadian rhythms. Moreover, if one were to cross such strain with a frq7 allele, my prediction is that the expression of GlnRS in the absence of adv-1 will look indistinctively circadian (particularly aided by the overall higher amplitude of rhythms in frq7) and with a longer period.

5.
" GlnRS rhythms were less robust in Δadv-1" In the discussion, the authors are less categoric about the effect of adv-1 and don't talk about arrhythmicity, while in table 2(and Fig 4) they use the latter concept.

6.
Is the work clearly and accurately presented and does it cite the current literature? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Circadian rhythms, Neurospora genetics and gene expression