Ribosome rescue factor PELOTA modulates translation start site choice and protein isoform levels of transcription factor C/EBPα

Translation initiation at alternative start sites can dynamically control the synthesis of two or more functionally distinct protein isoforms from a single mRNA. Alternate isoforms of the hematopoietic transcription factor CCAAT-enhancer binding protein α (C/EBPα) produced from different start sites exert opposing effects during myeloid cell development. This alternative initiation depends on sequence features of the CEBPA transcript, including a regulatory upstream open reading frame (uORF), but the molecular basis is not fully understood. Here we identify trans-acting factors that affect C/EBPα isoform choice using a sensitive and quantitative two-color fluorescence reporter coupled with CRISPRi screening. Our screen uncovered a role for the ribosome rescue factor PELOTA (PELO) in promoting expression of the longer C/EBPα isoform, by directly removing inhibitory unrecycled ribosomes and through indirect effects mediated by the mechanistic target of rapamycin (mTOR) kinase. Our work provides further mechanistic insights into coupling between ribosome recycling and translation reinitiation in regulation of a key transcription factor, with implications for normal hematopoiesis and leukemiagenesis.


Introduction Results
A fluorescent reporter measures CEBPA translation start site choice To measure translation start site selection on CEBPA, we developed a dual color reporter that converted the two translational isoforms of C/EBPα into two distinct fluorescent proteins produced from the same mRNA. Because the shorter isoform is an N-terminal truncation of the longer isoform, we could not simply mark each isoform with its own fluorescent protein. Instead, we fused the fast-folding, red fluorescent protein mScarlet-I 45 to the shared C-terminus, such that it would be expressed in both isoforms. Red fluorescence would therefore serve as a proxy for total protein abundance. We then inserted a green fluorescent protein into the long-isoform-specific N-terminal extension so that green fluorescence reported specifically on long isoform abundance. Thus, the ratio of green to red fluorescence measures the relative abundance of the two translational isoforms ( Figure 1C): green/red ∝ expression of long isoform expression of long + short isoform The endogenous N-terminal extension on the long isoform of C/EBPα is substantially shorter than a fluorescent protein and its mRNA sequence may contain regulatory information. To minimize disruptions to the organization and regulation of the transcript, we encoded only the short (16 amino acid) fragment of the split, self-complementing green fluorescent protein mNeonGreen2 (mNG2 11 ) 46,47 in the CEBPA CDS between the long and short isoform start sites. Co-expression of our long isoform reporter with the larger fragment of mNeonGreen2 (mNG2 1−10 ) reconstituted green fluorescence ( Figure S1A). To increase the dynamic range and sensitivity of our reporter, we optimized the Kozak sequence around the uORF start codon to enhance short isoform expression. We further deleted the DNA binding domain of C/EBPα to mitigate any secondary transcriptional effects of overexpressing our reporter. We then stably integrated a single copy of this construct into the AAVS1 locus in a K562 human myeloid leukemia cell line stably expressing mNG2 1−10 to generate monoclonal reporter cell lines ( Figure S1B).
First, to verify that our reporter recapitulates the regulated choice between CEBPA translation start sites, we tested the effects of mutating the start codons in our reporter. Mutation of the uORF start codon abolished short isoform expression by eliminating reinitiation, and mutation of the long isoform start codon itself eliminated long isoform expression ( Figure 1D). Furthermore, the reporter with a mutation in the uORF start site-and thus exclusively long isoform expressionshowed a much higher ratio of green to red fluorescence relative to the wild-type (WT) reporter, confirming that fluorescence could be used to accurately monitor the isoform ratio ( Figure 1E). We further tested how fluorescence of the wild-type reporter changed under conditions that shift the CEBPA isoform ratio. Treatment with the allosteric mTOR inhibitor rapamycin reduces short isoform expression 14 . We recapitulated this effect in our system by treating our reporter cell line with the mTOR active-site inhibitor PP242 48,49 and observed an increase in the ratio between green and red fluorescence, relative to DMSO-treated cells, consistent with a shift towards long over short isoform expression ( Figure S1C). Thus, our fluorescent reporter measures changes in isoform ratio resulting from the choice between translation start sites. between long and short isoforms will change the green/red fluorescence ratio, similar to the effect we saw from mutating the uORF start codon. It is thus possible to select for these perturbations by fluorescence-activated cell sorting (FACS) 50,51 .
Because many translation factors are essential and mutants can provoke strong growth defects, we perturbed gene expression by CRISPR interference (CRISPRi) 52 , which produces strong partial loss-of-function phenotypes that allow uniform comparisons across essential and non-essential genes. We transduced our reporter cell line with four different, lentiviral CRISPRi sgRNA sublibraries that collectively comprised 57,900 guides targeting approximately 10,000 genes, in addition to 1,070 nontargeting control guides 53 . We carried out FACS to distribute transduced cells into four distinct bins depending on their green/red fluorescence ratio and quantified the relative frequency of each sgRNA across the four bins by high throughput sequencing (Figure 2A). Cells expressing a CRISPRi sgRNA that alters the isoform ratio should be unequally distributed across these four FACS bins ( Figure S2A).
Indeed, we identified dozens of sgRNAs that changed the C/EBPα isoform ratio. These sgRNAs were strongly shifted towards one side of the sorted population, despite the generally strong correlation in sgRNA abundance across different bins (Spearman's ρ = 0.75 − 0.76) (Figures 2B and S2B-D). We quantified the shift in isoform ratio for each sgRNA using a generalized linear model of its abundance in the four sorted bins ( Figures 2C and 2D). The vast majority of our 1,070 nontargeting sgRNAs showed no significant shift, demonstrating the specificity of our experimental design and analysis strategy ( Figure 2C, 2D, and S2B). Often, two or more independent sgRNAs targeting the same gene caused significant shifts, further arguing that out approach was robust.
Many translation factors emerged among the targets with the strongest and most significant changes in isoform ratio. These included sgRNAs against DENR and MCTS1, which greatly increased the green/red ratio (Figures 2B-D and S2C) [54][55][56][57] . Loss of DENR or MCTS1 reduces reinitiation after uORF translation in flies 38 , although work in human cells suggested that they primarily affect transcripts with extremely short, single-codon uORFs 58 . The shift towards long isoform production in these knockdowns is consistent with a defect in translation reinitiation at the short isoform start codon. Depletion of the eIF4G paralog eIF4G2/DAP5, also implicated in reinitiation 59,60 , caused a strong increase in green fluorescence as well ( Figure S3A). Interestingly, depletion of the initiation factor eIF2α, encoded by EIF2S1, actually decreased the green/red ratio, in contrast with the observation that increasing eIF2α availability causes a shift towards short isoform production 14 . More broadly, the targets that increased the long isoform fraction across our screen were enriched for gene ontology (GO) annotations for nucleic acid binding and various mRNA-related regulatory processes ( Figure 2E and F).
Depletion of the ribosome rescue factor PELO strongly reduced the green/red ratio, indicating a major shift towards short isoform production ( Figures 2B, 2D, and S2C). Notably, although PELO functions in translation 43,61,62 , it was not previously linked to uORF-mediated regulation, suggesting a distinct and perhaps CEBPA-specific role. Furthermore, PELO is homologous to the peptide release factor encoded by ETF1 that acts in normal translation termination 63 , and two sgRNAs targeting ETF1 showed a similar but weaker shift towards short isoform translation ( Figure  S3A). These results suggested that termination and recycling-perhaps after uORF translationcould affect start site choice and thus C/EBPα isoform ratios.

Reinitiation and ribosome rescue factors control CEBPA start site choice
We selected several genes with known roles in translation and RNA biology for individual validation. Two independent, clonal cell lines expressing an sgRNA against DENR both had higher green/red fluorescence ratio than cells expressing a nontargeting control sgRNA, in agreement with our results from flow sorting and sequencing; we saw similar results in two independent clones expressing sgRNAs against DAP5 (Figures 3A and S4A). We also recapitulated a lower green/red ratio in two clonal cell lines expressing an sgRNA against PELO ( Figures 3A and S4A). More broadly, we saw a strong correlation (r = 0.96) between flow cytometry measurements and FACS enrichment across a collection of seven other sgRNAs, each of which shifted the green/red ratio in the direction expected from screening results ( Figure 3B).
Short isoform expression depends primarily on downstream reinitiation after uORF translation. In general, ribosomes must be recycled after translation termination in order to prepare them for a new round of initiation. Previous work has shown that the DENR/MCTS1 heterodimer can promote recycling of 40S ribosomes in vitro 64 . More recently, this recycling activity has been linked with reinitiation after uORF translation; DENR/MCTS1 appear to remove tRNAs from post-termination ribosomal complexes, thereby allowing 40S ribosomes to recruit new initiator tRNA and continue scanning 39,65 . This role in post-termination recycling explains the effects of DENR depletion on CEBPA start site selection.
The close connection between recycling and reinitiation also suggests how PELO-which is implicated in ribosome rescue and recycling after aberrant termination-could affect the choice between start sites. We then set out to further investigate the link between rescue, recycling and reinitiation. We first confirmed that the isoform shift we saw with sgRNAs targeting PELO indeed arose due to PELO depletion. The strongest sgRNA against PELO greatly reduced PELO protein levels ( Figure 3C). To rescue this PELO depletion, we overexpressed PELO from the strong, constitutive EEF1A promoter. The rescue construct also contained a drug-controlled destabilization domain (DD) that is stabilized in the presence of the small molecule Shield1 ( Figure S5A) 66 . We stably integrated either the PELO rescue construct, or an inactive HaloTag control, into our reporter cell line ( Figure S5B). Next, we transduced either a strong PELO sgRNA or a nontargeting control into each of these two cell lines and treated cells with Shield1. We found that PELO overexpression-but not HaloTag overexpression-completely rescued the PELO knockdown phenotype ( Figure 3D) and mitigated the substantial cell viability defect caused by PELO depletion as well ( Figure 3E). To confirm that the effect of PELO was not an artifact of our modified uORF start context, we recapitulated PELO knockdown and rescue with a reporter harboring the native Kozak sequence around the uORF start codon ( Figures S5C and S5D).
While it seemed likely that PELO depletion affected start site selection, we wanted to exclude the possibility that it had isoform-specific, post-translational effects on protein stability. To do so, we engineered a cell line containing a variant CEBPA reporter fused to a C-terminal, destabilizing PEST sequence, ensuring the rapid turnover of both isoforms. In this cell line, we again recapitulated the reduction in the green/red fluorescence ratio upon sgRNA-mediated PELO knockdown, arguing that this shift was not mediated by differences in protein half-life between the two isoforms ( Figure S5E). Instead, it appears that PELO plays an uncharacterized role in regulating translation start site selection on CEBPA.

PELO effects on CEBPA translation depend on uORF length
As PELO has not previously been implicated in uORF-mediated regulation, we next asked whether its effect on the relative abundance of the two C/EBPα isoforms depended on the uORF. We generated cell lines expressing either a reporter variant with a mutation inactivating the uORF start codon (Figure 1D), or a reporter with a short, unstructured 5 UTR containing no uORFs in place of the endogenous 5 UTR of CEBPA. Eliminating the uORF start codon or replacing the whole 5 UTR reduced short isoform expression, as reflected in the higher green/red fluorescence ratio observed in these cell lines ( Figure 4A). The effects of DENR and DAP5 knockdown were weakened or eliminated in these two reporters, which should no longer support reinitiation. The effects of PELO knockdown were likewise greatly attenuated in variants without uORFs; PELO depletion did not reduce the high green/red ratio ( Figure 4B). These observations suggest that the presence-and translation-of the CEBPA uORF is required for the change in isoform usage induced by PELO on our reporter.
We next considered the distinctive features of the CEBPA uORF. The length of the uORF and the short, 7 nucleotide separation between the end of the uORF and the long isoform start codon are conserved across CEBPA homologs, although their sequence varies ( Figure 4C). We thus tested whether uORF length impacted the effect of our knockdowns by generating stable two color reporter cell lines encoding the CEBPA uORF with varying lengths that still preserved the distance between the uORF stop codon and main CDS start codon. Shortening the uORF to 9 nucleotides reduced the green/red ratio, suggesting a shift towards short isoform production, in line with the general observation that shorter uORFs confer higher reinitiation probability 67 . In contrast, increasing the length of the uORF to 30 nucleotides increased the green/red ratio, indicating less efficient reinitiation ( Figure 4D).
When we introduced sgRNAs targeting PELO into each of these reporter lines, we found that the effect of PELO knockdown was most diminished on the shortest, 9 nucleotide uORF and was unchanged in the 30 nucleotide uORF reporter relative to the wild type, 18 nucleotide uORF reporter. In contrast, DENR knockdown produced a similar effect on the green/red ratio across all reporter lines ( Figure 4E). These results suggest that unlike DENR, PELO loss is especially sensitive to the relative positioning of the uORF start and stop codons.

PELO knockdown decreases long isoform expression
Our two-color reporter provides a sensitive measure of changes in the isoform ratio, but does not distinguish whether PELO knockdown reduces long isoform expression or increases short isoform expression. To delineate between these possibilities, we expressed a third fluorescent protein that would serve as a normalizing control and allow us to quantify the absolute abundance of each CEBPA isoform. We chose the infrared fluorescent protein iRFP670 68,69 , which is spectrally distinct from mNeonGreen2 and mScarlet-I, enabling simultaneous quantification of all three fluorescent proteins by flow cytometry, and expressed it using the constitutive EEF1A promoter. We calibrated our fluorescence measurements using a reporter with a mutation in the uORF start codon that expresses exclusively the long isoform, as well as a reporter that expresses only the short isoform ( Figures 1D and 5A).
We then used our calibrated fluorescent measurements to characterize the effect of several sgRNA knockdowns on absolute isoform abundance. We first confirmed that our targeted sgRNA knockdowns did not significantly impact the levels of our iRFP670 normalizer reporter ( Figure S6A). Consistent with their roles in promoting reinitiation at the short isoform start codon, depletion of DENR or MCTS1 notably reduced red fluorescence with no significant change in normalized green fluorescence, and thus no difference in long isoform levels. We observed a similar reduction in short isoform expression in DAP5 knockdown cells, in agreement with its proposed role in reinitiation. In PELO knockdown cells, we observed a decrease in long isoform expression while short isoform abundance was largely unaffected ( Figures 5B and S6B), implying that PELO normally promotes long isoform expression.
We next investigated the interactions between these distinct effects on long and short isoform translation. We knocked down either DENR or DAP5 in combination with PELO and compared these effects with single gene knockdown using a dual-sgRNA expression vector 70 (Table S1). The effect of depleting both DENR and PELO was additive-double knockdown reduced short isoform expression to the same extent as DENR knockdown alone, and long isoform expression to the same degree as PELO single knockdown. Interestingly, depleting DAP5 and PELO together somewhat weakened both the PELO-dependent loss of the long isoform as well as the DAP5 -dependent loss of short isoform expression ( Figures 5C and S6C). Nonetheless, the lack of strong epistasis argues that PELO is not directly affecting reinitiation.

PELO depletion increases CEBPA uORF translation and activates mTOR
To directly measure the translational effects of PELO depletion on our reporter, and across the transcriptome, we performed ribosome profiling 71,72 in our reporter cell line transduced with either the top scoring PELO sgRNA or a nontargeting sgRNA. Biological triplicates of the same sgRNA treatment correlated very well (p > 0.99) and showed clear, sgRNA-specific differences ( Figures S7C and S7D). We saw the characteristic accumulation of footprints in the 3 UTR in our PELO knockdown that correspond to unrecycled, vacant 80S ribosomes ( Figure S7A), as has been previously reported in both yeast and humans 43,44 . We further observed a striking increase in ribosome occupancy in the 5 UTR of our CEBPA reporter in PELO depleted cells relative to our control. These included footprints on the CEBPA uORF, indicative of increased uORF translation in PELO knockdown. We also observed a surprising accumulation of footprints that mapped to the long isoform start codon. While these footprints could be derived from ribosomes initiating at the long isoform start codon, our fluorescence measurements indicate that PELO depletion reduces long isoform production. Alternately, these footprints could originate from vacant ribosomes that accumulate after uORF termination, analogous to the unrecycled ribosomes that are enriched in 3 UTRs after main ORF termination as a consequence of reduced PELO levels. Greater persistence of these unrecycled ribosomes could occlude the long isoform start codon in PELO knockdown. They could also enhance uORF translation by stalling scanning, pre-initiation complexes at the uORF start codon, leading to a self-reinforcing situation where ribosomes that terminate after uORF translation favor subsequent uORF translation rather than long isoform translation. As this self-reinforcing effect depends on the precise position of the post-termination ribosome relative to the uORF start codon, it also explains the uORF length dependency of the PELO knockdown phenotype ( Figure 4E). Overall, these results support a model in which PELO loss leads to enhanced uORF translation at the expense of long isoform expression ( Figures 6A and S7B).
In addition to these CEBPA-specific effects, we observed a number of translational changes across the transcriptome. We computed translation efficiency (TE) as the ratio of ribosome footprint abundance to matched mRNA abundance. Overall, 248 genes displayed a significant (FDRadjusted p ≤ 0.05) TE difference in our PELO depletion ( Figure 6B). Among the genes with the strongest increase in TE, 32% (81 genes) were 5 terminal oligopyrimidine (TOP) motif containing mRNAs 73 primarily encoding ribosomal and ribosome-associated proteins ( Figure 6C). Translational upregulation of 5 TOP mRNAs is a hallmark of mTOR activation. Indeed, previous work in human fibroblasts and in mouse models also observed a marked translational enhancement of mTOR regulated transcripts in PELO knock-outs 74,75 . mTOR activation has broad ranging effects on protein synthesis and acts primarily on translation initiation by altering availability of the cap-binding protein eIF4E 76,77 . In fact, previous work has shown that rapamycin-induced mTOR inhibition favors C/EBPα long isoform expression by decreasing eIF4E availability 14 , raising the possibility that mTOR activation explains the reduced long isoform expression in PELO knockdown. We thus wanted to ask whether PELO affects C/EBPα translation above and beyond mTOR-mediated changes.
Despite the known effect of mTOR activity on CEBPA translation, genes from this pathway did not stand out in our screen. We identified a modest but significant effect from just one sgRNA 9 . CC-BY 4.0 International license available under a (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 The copyright holder for this preprint this version posted January 17, 2023. targeting MTOR itself, and no significant effects from sgRNAs targeting mTOR regulators RHEB or TSC1 ( Figure S7E). Individual knockdown of MTOR led to a slight increase in the green/red ratio, much smaller than the change seen in PELO knockdown, consistent with the weak phenotype in our screening data. We likewise saw that targeted knockdown of the mTORC1 activator RHEB did not change the fluorescence phenotype ( Figures 6D and S7E), nor did an individual sgRNA against the negative mTOR regulator TSC1 ( Figures S7E and S7F).
CRISPRi knockdown produces only partial loss of function phenotypes, and mTOR activity is modulated by feedback at many levels that might buffer the effects of genetic perturbation. We thus wanted to confirm that MTOR knockdown reduced phosphorylation of mTOR targets. Indeed, CRISPRi against MTOR reduced phosphorylation of 4EBP1 and almost abolished phosphorylation of RPS6, an indirect translation-related target, confirming that MTOR knockdown reduces mTOR activity ( Figure S7G). We also observed that PELO knockdown increases mTOR activity ( Figure  S7G).
We then depleted PELO in conjunction with either MTOR or the mTORC1 activator RHEB. Critically, the PELO depletion phenotype was unaffected by simultaneous knockdown of either MTOR or RHEB, arguing that it does not depend on increased mTOR activity. We further tested how the PELO knockdown phenotype was affected by the potent mTOR active-site inhibitor PP242, which has a stronger effect than MTOR knockdown (Figures 6D and S1C). We transduced our reporter line with either a PELO sgRNA or a nontargeting control and treated these cell lines with PP242. Consistent with the strong effect of PP242 in other settings, we saw that it substantially increased the green/red ratio in PELO knockdown cells ( Figure 6E). Nonetheless, PELO knockdown still reduced the green/red ratio in the context of PP242 treatment relative to a nontargeting control, consistent with a direct, mTOR-independent role for PELO in start site selection. Further analysis indicated that PP242 treatment alone enhanced long isoform expression at the expense of short isoform synthesis, with little change in total abundance ( Figure 6F). When PELO was depleted, PP242 treatment enhanced long isoform production but did not restore total protein levels relative to control cells ( Figure 6F). This again suggests that lack of PELO directly impairs long isoform synthesis-perhaps by physical obstruction by unrecycled ribosomes-an effect that cannot be suppressed by mTOR inhibition.

Discussion
We survey the trans-acting factors that control the choice between alternate translation start sites that produce opposing isoforms of the key hematopoietic transcription factor C/EBPα. Known reinitiation factors DENR/MCTS1 and DAP5 play substantial roles in promoting short isoform expression, supporting uORF-dependent reinitation as the mechanism for translation from the downstream start codon. We also found that loss of ribosome rescue factor PELO, which has no described role in translation reinitiation or uORF-mediated regulation, reduces C/EBPα long isoform expression. Our work thus reveals an unexpected link between ribosome rescue and uORFmediated translational regulation. We propose that long isoform initiation is blocked by unrecycled ribosomes that accumulate after uORF translation, providing a mechanistic connection between ribosome rescue and start site selection.
Both PELO and DENR/MCTS1 have established molecular functions at the end of translation. The impact of depleting these factors on CEBPA start site choice suggests that the fate of ribosomes after uORF translation controls downstream translation. The DENR/MCTS1 heterodimer recycles 40S subunits after termination, and this activity is important for subsequent reinitiation in many situations, including ATF4 translation 38,39,56,58 . The CEBPA uORF may rely on DENR because it contains a Leu codon in the penultimate codon position, which confers a particularly strong dependency on DENR for post-termination tRNA eviction 39,64 .

10
. CC-BY 4.0 International license available under a (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 The copyright holder for this preprint this version posted January 17, 2023. ; While PELO is also associated with ribosome recycling 41,42 , loss of PELO affects long isoform initiation specifically and so it does not seem to prepare ribosomes for reinitiation. Instead, in keeping with its function in other contexts, PELO likely removes vacant, un-recycled ribosomes that would otherwise impede subsequent rounds of translation initiation on the transcript. The conserved length and position of the CEBPA uORF, near the long isoform start codon, is expected to enhance the consequences of a post-termination stall, because an 80S ribosome that is not released after uORF translation would be positioned over the long isoform start codon, thereby blocking its expression. The specific positioning of this stalled post-termination ribosome could also have an additional effect: because the combined 25-nucleotide length of the uORF and intercistronic region is nearly the size of a 28-nucleotide elongating ribosome footprint, incoming pre-initiation scanning complexes would be queued behind the stalled 80S and positioned near the uORF start codon, enhancing uORF translation. We found that a variant reporter with a shorter uORF was less responsive to PELO depletion, consistent with a requirement for the conserved spacing of the CEBPA uORF to strengthen the effects of unrecycled ribosomes on both uORF and long isoform initiation. This effect of post-termination complexes is reminiscent of other regulatory paradigms where translational stalling promotes upstream initiation 27,78-80 .
The CEBPA 5 UTR is highly structured in addition to containing a uORF, making it a prime target for DAP5 dependence as well 59 . While the molecular details of DAP5 function are not clear, its general role in downstream CDS translation in the context of structured 5 UTRs is consistent with its importance for short isoform translation on CEBPA. It is also notable that DAP5 -dependent translation requires efficient termination and recycling, again connecting the results of our screen to uORF translation termination.
We also found that PELO depletion activates the mTOR pathway, as seen previously in other systems 74,75 . Changes in mTOR activity have been shown previously to regulate translation start site usage on CEBPA 14 . Through a combination of genetic and chemical perturbations, we provide evidence for both mTOR dependent and independent effects of PELO depletion. Indeed, impaired ribosome recycling after PELO depletion may reduce translational capacity 81 , activating mTOR in a compensatory response. The mechanism linking PELO depletion with mTOR activation is unknown, but may involve the direct association of mTORC1 with ribosomes 82 or an imbalance between ribosome availability and protein biosynthesis capacity 81 .
Ribosome rescue and recycling activity are themselves dynamically regulated during erythroid development 44 . In both hematopoietic progenitor CD34+ cells and K562 cells, PELO is initially upregulated then gradually decreases during differentiation; PELO levels are greatly diminished in primary platelets and reticulocytes relative to proliferative, nucleated cells. Platelets and reticulocytes derive from the common myeloid progenitor, where CEBPA plays a central role in fate specification. In addition to erythroid and megakaryocyte/platelet lineages, the common myeloid progenitor gives rise to the granulocyte/monocyte progenitor, and CEBPA is required specifically for this cell fate decision 83 . The long isoform of CEBPA is critical for both granulocytic and monocytic maturation while overexpression of the short isoform blocks granulocyte differentiation 84 . Variations in PELO levels in these blood cell lineages may affect CEBPA isoform balance and by extension, myeloid cell fate commitment. Expression of PELO is also decreased in AML 85 , and our data suggest that this would favor the oncogenic, short isoform. These trends underscore the broader physiological and pathological impact of PELO and the ribosome rescue pathway on the regulation of hematopoeisis. scientific discussions. mNeonGreen2 plasmids were kind gifts of Siyu Feng. We also thank Jonathan Weissman's lab for the K562 CRISPRi cell line. Hector Nolla, Alma Nuguid Valeros and Kartoosh Heydari provided indispensable support at the Flow Cytometry Facility at UC Berkeley. We also thank the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley and the UC Berkeley DNA Sequencing Facility. This work was supported by the National Institutes of Health (www.nih.gov), grants DP2 CA195768 and R01 GM130996 (NTI) and shared instrumentation grant S10 OD018174. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Declaration of Interests
N.T.I. declares equity in Tevard Biosciences and Velia Therapeutics.
12 . CC-BY 4.0 International license available under a (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

Materials and Methods
Plasmid and two color reporter construction All plasmids and primers used are listed in Table S1 and S2, respectively. All plasmids (with the exception of sgRNA expression vectors, see section) were generated by Gibson assembly 86 from amplicons made with primers indicated in Table S2.
In brief, to construct the CEBPA two color reporter, the mNG2 11 sequence was subcloned from a pCMV-mNG2 11 -H2B plasmid kindly gifted by Siyu Feng 47 and assembled into codon position 45 in the N-terminus of CEBPA. All Met codons in mNG2 11 were removed to avoid generating new in-frame start sites and substituted with either Val or Ile to preserve hydrophobicity. Similarly, an in-frame start codon at position 14 in CEBPA was mutated to ACA (∆2) to ensure that only the long and short start sites were used. Furthermore, the basic, DNA-binding domain was deleted (∆DBD) to suppress cell proliferation arrest caused by ectopic expression of CEBPA 14,87 and the Kozak sequence around the uORF start codon was optimized (gccgccATGg, as in Calkhoven et al. 14 ) to increase the dynamic range of our reporter readout. Hs CEBPA cDNA was amplified from the genome. Amplicons corresponding to the SFFV promoter, CEBPA 5'UTR, CEBPA-mNG2 11 and CEBPA-mScarlet-1XFlag were then introduced into the Sbf1 and Kpn1 cut sites of the pNTI620 vector.

Generation of stable two color reporter cell lines
The mNG2 1−10 fragment was a gift from Siyu Feng 47 and was transfected using TransIT-LTI Transfection Reagent (Mirus) and packaged with pNTI673 and pNTI674 (Table S1) in a HEK 293T Lenti-X cell line to generate lentiviral particles. mNG2 1−10 was then stably integrated into a polyclonal dCas9-KRAB CRISPRi K562 cell line 52 by lentiviral transduction. Dual color CEBPA reporter constructs (WT, ∆uORF start, ∆Long start, Short UTR, TableS1) were then stably integrated into this cell line by Cas9-mediated integration into the AAVS1 locus by simultaneous nucleofection of plasmids containing either targeting sgRNAS (AAVS1-T2 and AAVS1-T2, Table  S3 ) and spCas9 88 and selected using 1 µg/mL Puromycin (Invivogen) to generate stable integrants. All lines were subsequently monoclonally isolated.
Cells were harvested for flow cytometry analysis by centrifugation (1500 RPM for 5 minutes at room temperature) followed by resuspension in PBS supplemented by 1% FBS and 1mM HEPES. All analyses were done on a LSR Fortessa Analyzer (BD Biosciences). Cells were initially gated on forward (FSC) and side scatter (SSC) ( Figure S1A) and positive events were determined by a threshold based on negative (no stain) and single color control cells. Green, red and IRFP fluorescence was detected on the FITC (530/30 nM), PE-Texas Red (610/20 nM) and APC-Cy7 (780/60 nM) channels, respectively.

FACS-based CRISPRi screen
CRISPRi sublibrary screens were performed using four, compact BFP-tagged CRISPRi sublibraries containing 5 sgRNAs per TSS (Addgene, Cat#83971-3 and #83975) expressed in the pCRISPRi-v2 expression vector (Addgene, Cat#84832). Plasmid sublibraries were separately packaged in HEK 293T Lenti-X cells and transduced into the CRISPRi dual color reporter line at an MOI < 1 where the percentage of transduced cells by BFP expression after 2 days post-transduction was 20%-30%. At 2 days post-transduction, we performed fluorescence activated cell sorting (FACS) using an Aria Fusion (BD Biosciences) to select for cells expressing BFP. Cells with the highest (∼20%) BFP expression were collected and recovered in RPMI 1640 for 6 days post-FACS. Approximately 10 million cells were collected per sublibrary, maintaining an average sgRNA coverage of at least 500 cells per sgRNA.
At 6 days post-BFP selection, cells were again sorted using a FACS Aria Fusion based on the ratio of green/red fluorescence from our CEBPA dual color reporter line. Approximately 40 million cells per sublibrary transduction were sorted into four, distinct green/red bins (∼20-25% of cells in each bin), with each bin containing ∼8-10 million cells to ensure an average sgRNA/cell coverage of at least 500. Genomic DNA was immediately harvested from these cells using the DNeasy Blood and Tissue kit (Qiagen, 69504) and sgRNA fragments were isolated by SbfI (New England Biolabs) restriction digestion and Ampure bead size selection then amplified by PCR for deep sequencing as described in 53 . The sgRNAs were sequenced on an Illumina HiSeq-4000 using custom primers.

CRISPRi screen processing and data analysis
Sequencing reads were trimmed to remove adapter sequences using Cutadapt 90 and trimmed sgRNAs were counted using MAGeCK 91,92 . Raw sgRNA counts were then used as input to DE-Seq2 93 to calculate enrichment scores (Isoform Shift scores) in which each fluorescent bin (FR, NR, NG, FG) was represented as a numeric covariate in the linear model such that: bins = (−1, 0, 0, 1) This assumes that sgRNA counts in the FR and FG bins (at the extremes of the fluorescent ratio distribution) have a constant multiplicative change with respect to the middle (NR and NG) bins.
Gene Ontology analysis was performed using PANTHER 94-96 with background lists representing the genes targeted by each hCRISPRa-v2 sublibrary.

Individual validation of sgRNA-mediated phenotypes
Individual sgRNA expression vectors were cloned by first annealing complementary synthetic oligonucleotide sequences containing each sgRNA protospacer (Table S3)  CC-BY 4.0 International license available under a (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 The copyright holder for this preprint this version posted January 17, 2023. ; https://doi.org/10.1101/2023.01.16.524343 doi: bioRxiv preprint double-stranded annealed pair was then ligated into a BstXI/BlpI-digested pCRISPRi-v2 expression vector containing a BFP cassette. Each sgRNA expression vector was then packaged into lentiviruses in HEK 293T Lenti-X cells and were individually transduced into either two or three color CEBPA CRISPRi reporter lines at a MOI < 1, resulting in ∼20-30% infected cells by BFP expression. Cells were sorted on BFP 2 days post-transduction to select for sgRNA expression and allowed to recover in RPMI 1640 for 6 days. Reporter expression was then measured by flow cytometry.

PELO re-expression rescue assays
The HaloTag and PELO CDS was fused to the FKBP12 destabilizing domain (DD) to generate (N-terminal) DD-HaloTag and PELO-DD (C-terminal) constructs and were stably integrated into K562 CRISPRi dual color reporter lines by Sleeping-beauty transposition 89 (Sleeping-beauty expression vector, Table S1). Polyclonal cells were selected using 200 µg/mL Hygromycin (Invivogen). Single sgRNAs were then packaged and transduced (as described above) into these cell lines and sorted for BFP-tagged sgRNA expression. Cells were then treated with 1 µM Shield1 (Takara Bio Cat#632189) 5 days post-BFP sort then harvested for flow cytometry and Western blot analysis 3 days post-Shield1 treatment. Because of the incomplete destabilization of the PELO-DD construct (due to the necessity of placing the DD-tag at the C-terminus), we were unable to use a noninduced condition as a point of comparison ( Figure S5A).

Ribosome profiling and RNA-sequencing
For ribosome profiling and matched RNA sequencing, K562 CRISPRi dual color reporter cells were first transduced with either a nontargeting sgRNA or the top scoring PELO sgRNA (see individual sgRNA knockdown validation) in triplicate and were grown in T150 flasks (Corning) for 6 days post-BFP selection. Cells (5.0x10 6 nontargeting sgRNA and 2.5x10 6 PELO sgRNA per replicate) were harvested as previously described 72 without addition of cycloheximide and a sample of the lysate was taken for RNA sequencing. Ribosome profiling was done as described in 72 .
For matched RNA sequencing, cells were harvested by phenol-chloroform extraction and processed according to the NEB Ultra II Directional RNA Sample Prep Kit (New England Biolabs, #E7760S).

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. CC-BY 4.0 International license available under a (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 The ribosome profiling and total RNA sequencing samples were sequenced on an Illumina NovaSeq instrument.

Ribosome profiling sequencing analysis
Ribosome profiling reads were first trimmed using Cutadapt and aligned to ribosomal RNA (rRNA) and transfer RNA (tRNA) references using Bowtie2 97 . The remaining reads were subsequently aligned to the transcriptome using STAR 98 . Transcriptome-based alignments were then filtered to exclude the first 15 and last 5 codons due to the accumulation of initiating and terminating ribosomes. Finally, footprint abundance was quantified from these alignments by RSEM 99 . Differential expression and translation efficiency analysis was conducted using DESeq2 93 .

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. CC-BY 4.0 International license available under a (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  25 . CC-BY 4.0 International license available under a (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  (E) Gene ontology (GO) terms associated with sgRNAs that were enriched in cells expressing more long isoform (a higher green/red ratio) in the Gene Expression sublibrary screen. Only sgRNAs with a FDR < 0.05 were included in the analysis. GO term analysis was performed using Fisher's exact test using the Bonferroni correction for multiple testing. Categories chosen represent the most statistically significant terms with a fold enrichment > 29. (F) As in (E), but with sgRNAs from the Cancer and Apoptosis sublibrary screen.

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. CC-BY 4.0 International license available under a (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 The copyright holder for this preprint this version posted January 17, 2023. ; https://doi.org/10.1101/2023.01.16.524343 doi: bioRxiv preprint   28 . CC-BY 4.0 International license available under a (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

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. CC-BY 4.0 International license available under a (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  (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 The copyright holder for this preprint this version posted January 17, 2023. ; containing mRNAs. (D) Median green and red fluorescence normalized to IRFP670 in stable cell lines expressing the wild type three color reporter transduced with indicated dual sgRNAs. (E) Median green/red fluorescence measurements in reporter cell lines transduced with the indicated sgRNAs. Cells were treated with either DMSO or 1 µM PP242 (Sigma-Aldrich) 6 days posttransduction. Cells were harvested and assayed by flow cytometry 48h post-drug treatment. (F) Flow cytometry measurements of median green and red fluorescence from (E), n = 3.

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. CC-BY 4.0 International license available under a (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 The copyright holder for this preprint this version posted January 17, 2023.

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. CC-BY 4.0 International license available under a (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  Figure 2: Validation of FACS-based CRISPRi sublibrary screens (A) Schematic representation of possible, functional outcomes of sgRNA-mediated knockdown on the two color reporter. Bin labels reflect green/red distribution. FR: far red; NR: near red; NG: near green; FG: far green. sgRNAs that are enriched in cells with higher green/red ratios (NG and FG) may be changing the ratio by shifting expression towards the long isoform. We consider this class of sgRNAs as positive regulators of short isoform expression. In contrast, sgRNAs that are enriched in cells with lower green/red ratios (NR and FR) may be changing the ratio by shifting isoform expression towards the short isoform. We consider this class of sgRNAs as negative regulators of short isoform expression. sgRNAs that are uniformly distributed across all bins either have no effect on isoform usage or affect both isoforms equivalently. We classify these sgRNAs as being neutral. (B) sgRNA read count distribution across all bins for 6 nontargeting sgRNAs from the Gene Expression sublibrary. (C) Comparison of sgRNA read counts between the Far Red (FR) and Far Green (FG) bins in the Gene Expression sublibrary (left) and Cancer and Apoptosis sublibrary (right). Individual sgRNAs against DENR, MCTS1 and PELO are highlighted. (D) Comparison of sgRNA read counts between indicated fluorescent bins in the Cancer and Apoptosis sublibrary (as in Figure 2D). Each point represents one distinct sgRNA.

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. CC-BY 4.0 International license available under a (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 The copyright holder for this preprint this version posted January 17, 2023. ; https://doi.org/10.1101/2023.01.16.524343 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (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 The copyright holder for this preprint this version posted January 17, 2023. ; https://doi.org/10.1101/2023.01.16.524343 doi: bioRxiv preprint of green/red fluorescence by flow cytometry in a two color reporter cell line harboring the native, uORF Kozak sequence (ctcgccATGc) stably expressing either a DD-HaloTag or PELO-DD fusion construct and transduced with either a nontargeting or a PELO sgRNA. Cells were transduced and treated as in (B). (D) Median green/red fluorescence measurements of the cell lines in (C), n = 3. (E) Distribution of green/red fluorescence by flow cytometry in a two color reporter cell line bearing a C-terminal PEST sequence transduced with either a nontargeting or PELO sgRNA. (F) Median green/red fluorescence measurements of PEST-fusion two color reporters in (E), n = 3.

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. CC-BY 4.0 International license available under a (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 The copyright holder for this preprint this version posted January 17, 2023.  Figure 5B. Fluorescent values were normalized to the median values in the negative, nontargeting control. (C) Flow cytometry measurements of median green and red fluorescence normalized to IRFP in stable cell lines expressing the wild type three color reporter transduced with indicated dual sgRNAs, n = 3, as in Figure 5C. Fluorescent measurements were normalized as in (B).

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. CC-BY 4.0 International license available under a (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 The copyright holder for this preprint this version posted January 17, 2023. ; https://doi.org/10.1101/2023.01.16.524343 doi: bioRxiv preprint Cancer and Apoptosis sublibrary (right). Individual sgRNAs against MTOR, RHEB, TSC1, TSC2 and RAPTOR are highlighted. (F) Flow cytometry measurement of three color reporter cell lines transduced with either a sgRNA against TSC1 or a nontargeting sgRNA. Median green/red ratio is displayed, n = 3 (left). Flow cytometry measurements of median IRFP670 fluorescence in stable cell lines expressing the three color reporter transduced with indicated sgRNAs, n = 3 (right). (G) Western blot of three color reporter cell lines transduced with the indicated dual sgRNAs as in Figure 6D. β-actin was used as a loading control. Replicates represent separate, independent transductions.

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. CC-BY 4.0 International license available under a (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 The copyright holder for this preprint this version posted January 17, 2023. ; https://doi.org/10.1101/2023.01.16.524343 doi: bioRxiv preprint