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Nuclear RNA Concentration Coordinates RNA Production with Cell Size in Human Cells

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bioRxiv preprint doi: https://doi.org/10.1101/2021.05.17.444432; this version posted May 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Nuclear RNA concentration coordinates RNA production with cell size in human cells Scott Berry1*, Micha Müller1, Lucas Pelkmans1* 1Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland. *Correspondence: [email protected], [email protected] Unlike its DNA template, RNA abundance and synthesis rates increase with cell size, as part of a mechanism of cellular RNA concentration homeostasis. Here, we study this scaling phenomenon in human cells by combining genome-wide perturbations with quantitative single-cell measurements. Despite relative ease in perturbing RNA synthesis, we find that RNA concentrations remain highly constant. Systems-level analysis indicates that perturbations that would lead to increased nuclear mRNA abundance result in downregulation of mRNA synthesis. This is associated with reduced levels of several transcription-associated proteins and protein states that are normally coordinated with RNA production in single cells, including RNA polymerase II (Pol II) itself. Acute shut-down of nuclear RNA degradation, elevation of nuclear mRNA levels, and mathematical modelling indicate that mammalian cells achieve RNA concentration homeostasis by an mRNA-based negative feedback on transcriptional activity in the nucleus. Ultimately, this acts to robustly scale Pol II abundance with cell volume and coordinate mRNA synthesis and degradation. INTRODUCTION et al., 2015; Sun et al., 2020), but causal evidence is lacking. Within a cell population, the number of mRNA transcripts of a given gene is highly variable between Control of RNA degradation has thus far not been cells (Raj and Oudenaarden, 2008). Yet much of this implicated in cell size-scaling, however studies in variability can be explained by variation in cell size – yeast have revealed that disruption of RNA with larger cells containing more transcripts (Battich degradation tends to result in compensatory et al., 2015; Padovan-Merhar et al., 2015). Such decreases in RNA synthesis rates, and vice versa, ‘scaling’ of mRNA abundance with cell size is highly that disrupting transcription leads to decreases in conserved and is underpinned by increasing RNA degradation rates (Haimovich et al., 2013; Sun transcript production rates with cell size, while et al., 2012, 2013). In yeast, this ‘buffering’ degradation rates remain constant (Ietswaart et al., mechanism was reported to involve direct 2017; Kempe et al., 2015; Padovan-Merhar et al., (Haimovich et al., 2013) or indirect (Sun et al., 2013) 2015; Sun et al., 2020; Zhurinsky et al., 2010). transcriptional regulation by the cytoplasmic 5ʹ-3ʹ However, despite these consistent observations exonuclease Xrn1. In mammalian cells, similar across eukaryotes, we still lack a mechanistic systematic studies of mRNA buffering are lacking understanding of how cells control global RNA (reviewed in (Hartenian and Glaunsinger, 2019)), production, and how this is coordinated with cell size. although there have been reports of mRNA The predominant mechanistic hypothesis is the stabilisation upon transcriptional inhibition (Helenius ‘limiting factor model’, which supposes that cells et al., 2011; Slobodin et al., 2020). In contrast to contain a factor that is limiting for transcription and yeast, however, both acceleration (Abernathy et al., whose abundance is closely coupled to cell size 2015; Gilbertson et al., 2018) and disruption of (Marguerat and Bähler, 2012). If this limiting factor is cytoplasmic RNA degradation (Lee et al., 2012; localised to a cellular structure that does not scale Singh et al., 2019) have been associated with with cell size (e.g. DNA), then its local concentration transcriptional repression. The role of the cytoplasm on that structure will be proportional to cell size, in mRNA buffering therefore remains unclear in despite having constant global concentration mammalian cells. Moreover, in both yeast and (Padovan-Merhar et al., 2015). It has been mammalian systems, studies of mRNA buffering suggested that RNA Polymerase II (Pol II) itself is the have focused on cell population measurements and limiting factor (Lin and Amir, 2018; Padovan-Merhar have not considered effects of cell size changes in 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.17.444432; this version posted May 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. perturbations, complicating the interpretation of their to cell volume, and volume typically still accounted for effects. the majority of transcript abundance variation (Figure S1I-L). Moreover, mean mRNA abundance in Since both buffering and size-scaling contribute to perturbations was well predicted by the change in mRNA concentration homeostasis, they may be part volume (Figure S1M) and normalising spot count by of the same mechanism, but whether this is the case, volume at the single-cell level led to overlapping and how this works is unknown. To study these distributions for most genes (Figure 1B, S1N). Cell phenomena together, we here combine genome- volume is therefore a dominant source of wide genetic perturbation screening with multiplexed heterogeneity in mRNA abundance in cell quantitative measurements of single cells. Crucially, populations, and its perturbation leads to precise this enables us to connect effects seen in adaptation of transcript abundance to maintain gene- perturbations with naturally varying properties of specific mRNA concentration. unperturbed cells. We uncover hundreds of perturbations that affect global RNA synthesis rates in single cells, including pathways not previously RNA production rates are coupled to cell cycle implicated in transcriptional control. However, in most and cell volume cases we find that RNA concentration is not disrupted. Systems-level analysis of the genes RNA abundance is determined by production and involved together with detailed characterisation of degradation rates. However, inferring gene-specific molecular phenotypes at the single-cell level suggest mRNA production rates in single cells is technically a model in which transcription rates are negatively challenging and often requires assumptions regulated by nuclear mRNA concentration. We (Ietswaart et al., 2017; Padovan-Merhar et al., 2015; propose that the activity, and ultimately abundance, Sun et al., 2020). Because size-scaling is a of RNA Pol II is determined by this mRNA-based transcriptome-wide phenomenon, we measured bulk feedback, to enable robust mRNA concentration RNA production in single cells in situ using metabolic homeostasis in human cells. pulse labelling with 5-ethynyl uridine (EU) (Jao and Salic, 2008; Padovan-Merhar et al., 2015; Shah et al., 2018) in combination with measurement of cell RESULTS volume and stains to assign cells to G1/S/G2 cell- cycle phases (Figure 1C-D, S2A-J). In two cell lines Cell size perturbation leads to precise adaptation (HeLa, 184A1) and in primary human keratinocytes, of mRNA abundance EU incorporation increased with cell volume (Figure In unperturbed human cells, mRNA abundance S2I), in agreement with previous results (Padovan- scales with cell size (Figure S1A) (Battich et al., 2015; Merhar et al., 2015). However, including cell-cycle Kempe et al., 2015; Padovan-Merhar et al., 2015). To information revealed that G2 and S-phase cells investigate if mRNA concentrations are robustly showed higher EU incorporation than G1 cells of the maintained upon cell volume perturbation, we same volume (Figure 1D, S2J). Furthermore, HeLa established a method of cell volume measurement cells showed an additional increase during S-phase compatible with high-throughput branched-DNA (Pfeiffer and Tolmach, 1968) – above the level seen (bDNA) smFISH (Figures S1B-H, STAR methods) in G2. While these cell cycle effects are interesting and measured cytoplasmic transcript abundance for and suggest differences compared to yeast (Voichek 14 size-scaling genes (Battich et al., 2013) in et al., 2016), we here focus on cell-size scaling of EU populations of HeLa cells. Cell size was perturbed incorporation – using precise cell cycle information to using siRNA-mediated knockdown of GRIP2 and exclude changes to DNA template abundance. SBF2, genes which are not known to affect EU is incorporated into all major RNA species by transcription but whose knockdown results in smaller RNA polymerases I, II, and III (Jao and Salic, 2008). and larger cells, respectively (Berchtold et al., 2018) To evaluate the contribution of ribosomal RNA (�" = 1.0 pL, �" = 2.3 pL, -� = 4.9pL). GRIP2 Scrambled SBF2 (rRNA) to EU incorporation, we treated cells with the Average mRNA abundance was dramatically RNA Pol I inhibitor CX-5461 (Drygin et al., 2011), reduced in smaller cells (GRIP2 RNAi) and increased which resulted in elimination of EU incorporation in in larger cells (SBF2 RNAi) (Figure 1A-B, S1I). the nucleolus – the site of rRNA transcription (Figures However, mRNA abundance remained proportional 1E, S2G-H). 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.17.444432; this version posted May 17,
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