A human ESC-based screen identifies a role for the translated lncRNA LINC00261 in pancreatic endocrine differentiation

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Abstract

Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Using a human embryonic stem cell (hESC)-based pancreatic differentiation system, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for pancreatic endocrine cell development. However, deletion of LINC00261 diminishes insulin⁺ cells, in a manner independent of the nearby TF FOXA2. One-by-one disruption of each of LINC00261's open reading frames suggests that the RNA, rather than the produced microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs during hESC pancreatic differentiation and provides a blueprint for dissection of their coding and noncoding roles.

Introduction

Defects in pancreatic endocrine cell development confer increased diabetes risk later in life (Bakhti et al., 2019). Therefore, a detailed understanding of the factors that orchestrate endocrine cell differentiation is highly relevant to human disease. Many of the molecular mechanisms that underlie the formation of pancreatic endocrine cells have been defined (Romer and Sussel, 2015; Schiesser and Wells, 2014). However, despite some evidence that long noncoding RNAs (lncRNAs) are important for proper development and function of pancreatic beta cells (Arnes et al., 2016; Morán et al., 2012; Wong et al., 2019), a systematic functional assessment of the noncoding transcriptome during pancreas development is lacking.

Most lncRNAs with to date demonstrated roles in the regulation of fundamental developmental processes are active in the cell's nucleus (Daneshvar et al., 2016; Jiang et al., 2015; Klattenhoff et al., 2013; Kurian et al., 2015; Lin et al., 2014; Luo et al., 2016; Ramos et al., 2015). However, a large proportion of lncRNAs is predominantly cytosolic (Cabili et al., 2015; van Heesch et al., 2014), and the functional relevance of these lncRNAs has remained unexplored in the context of human development. It is now widely accepted that many cytosolic lncRNAs possess short, ‘non-canonical’ open reading frames (sORFs) that are translated (Bazzini et al., 2014; Makarewich and Olson, 2017; Ruiz-Orera et al., 2014). What fraction of these non-canonical ORFs is functional, and whether sORF translation serves a pure regulatory purpose or results in the production of stable microproteins, remains an active topic of debate (Levy, 2019; Ruiz-Orera et al., 2018). Since high rates of conservation have historically been employed for the identification and annotation of canonical protein coding sequences (Lin et al., 2011; Mudge et al., 2019), a primary reason for doubting the protein-coding capacity of sORFs in presumed lncRNAs is their generally poor sequence conservation across species. To address these questions, several recent studies have systematically assessed the biological activity of newly discovered sORFs, revealing that many produce evolutionary young microproteins with roles across cellular organelles and processes, and a subset being essential for cell survival (Chen et al., 2020; Martinez et al., 2020; Prensner et al., 2020; van Heesch et al., 2019). This previously unrecognized coding capacity of supposedly noncoding RNAs illustrates their functional diversity and has called into question the noncoding classification of some lncRNAs. Thus, there is a need for careful investigation and dissection of any gene's coding and noncoding functions.

LncRNAs, translated or fully noncoding, are not randomly distributed in the genome but are frequently located close to, and coregulated with, canonical protein-coding genes in cis (Luo et al., 2016; Neumann et al., 2018; van Heesch et al., 2019). For example, the lncRNAs DIGIT (also known as GSC-DT) and Gata6as (also known as lncGata6 or GATA6-AS1) have been reported to enhance expression of divergently expressed endoderm regulators Goosecoid (GSC) and Gata6, respectively (Daneshvar et al., 2016; Luo et al., 2016; Neumann et al., 2018). Similarly, the Pax6-associated lncRNA Paupar promotes pancreatic islet alpha cell formation through the alternative splicing of Pax6 transcripts in mice (Singer et al., 2019). Furthermore, LINC00261 (also known as DEANR1) and its neighboring TF FOXA2 are both induced in endoderm formation, during which LINC00261 has been proposed to positively regulate FOXA2 expression (Jiang et al., 2015). However, whether such cis-acting lncRNAs are translated and may exert cytosolic functions through trans-acting, microprotein-dependent mechanisms relevant for endoderm and pancreas development is not known.

In this study, we classified lncRNAs based on their dynamic regulation, subcellular localization, and translation in a hESC differentiation system that recapitulates in vivo pancreas development. Next, we used this classification to prioritize select dynamically regulated and highly translated lncRNAs for deletion in hESCs, followed by extensive phenotypic characterization across multiple intermediate states of pancreas development. Nine out of the ten selected lncRNAs were not essential for pancreatic development and, despite their vicinity to lineage-determining TFs, none of these lncRNAs regulated the expression of these TFs in cis.

The deletion of one lncRNA, LINC00261, impaired human endocrine cell development and led to a significant reduction in the number of insulin-producing cells. Contrary to previous studies of LINC00261 knockdown hESCs (Jiang et al., 2015), deletion of LINC00261 had no effect on the expression of nearby TF FOXA2 or other proximal genes, suggesting control of endocrine cell formation through a trans- rather than cis-regulatory mechanism. LINC00261 was among the most highly translated lncRNAs based on ribosome profiling (Ribo-seq) and produced multiple microproteins with distinct subcellular localizations upon overexpression in vitro. To systematically assess LINC00261's coding and noncoding functions, we separately introduced frameshift mutations into each of seven identified LINC00261 sORFs. However, rigorous phenotypic characterization revealed no apparent consequences of loss of each of the seven LINC00261-sORF-encoded microproteins on endocrine cell development. Our comprehensive assessment of functional lncRNA translation identified a likely trans-regulatory role for LINC00261 in endocrine cell differentiation that appears to be independent of the seven microproteins that were individually deleted. With this detailed investigation we provide a blueprint for the proper dissection of a gene's coding and noncoding roles in a human disease-relevant system.

Results

LncRNAs and nearby lineage-determining transcription factors exhibit dynamic coregulation during pancreas development

To identify lncRNAs involved in the regulation of pancreas development, we profiled RNA expression at five defined stages of hESC differentiation toward the pancreatic lineage: hESCs (ES), definitive endoderm (DE), primitive gut tube (GT), early pancreatic progenitor (PP1), and late pancreatic progenitor (PP2) (Figure 1A). While some lncRNAs were constitutively expressed (n = 592; 25.3%), the majority showed dynamic expression patterns (n = 1745; 74.7%), being either strongly enriched in (n = 874; 37.4%) or specific to (n = 871; 37.3%) a single developmental intermediate of pancreatic lineage progression (Figure 1B and Figure 1—source data 1A). The expression of many of these dynamically regulated lncRNAs correlated with that of proximal coding genes (Figure 1—figure supplement 1A–D and Figure 1—source data 1B,C), further exemplified by a subset of lncRNAs that was specifically coregulated with the key endodermal and pancreatic TFs GATA6, FOXA2, PDX1, and SOX9 (Figure 1C,D). The expression coregulation of these lncRNA-TF pairs is likely explained by a shared chromatin environment (Figure 1—figure supplement 1E–H), which raises the possibility that like the TFs, the function of the lncRNAs is also required for endoderm and pancreas development.

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LncRNA expression and regulation during pancreatic differentiation.

(A) Stages of directed differentiation from human embryonic stem cell (hESCs) to hormone-producing endocrine cells. The color scheme for each stage is used across all figures. (B) K-means clustering of all lncRNAs expressed (RPKM ≥ 1) during pancreatic differentiation based on their expression z-score (mean of n = 2 independent differentiations per stage; from CyT49 hESCs). (**C,D**) Left: Scatterplots comparing the expression of early (C) and late (D) expressed endodermal transcription factors (TFs) with the expression of their neighboring lncRNAs across 38 tissues. The dot color indicates the germ layer of origin of these tissues. Pearson correlation coefficients and p-values (t-test) are displayed. Right: Distribution of the Pearson correlation coefficients for each TF with all Ensembl 87 genes across the same 38 tissues. Dashed lines denote the correlation for the neighboring lncRNA, which for all lncRNAs shown is higher than expected by chance. See also Figure 1—figure supplement 1 and Figure 1—source data 1.

Figure 1—source data 1 Identification, regulation, and characterization of lncRNAs during pancreatic differentiation. (A) Gene expression during pancreatic differentiation (RPKM). (B) lncRNA-proximal TFs, by cluster in correlation heatmap (Figure 1—figure supplement 1C). (C) GO enrichment and KEGG pathway analysis for each cluster in the correlation heatmap (Figure 1—figure supplement 1D).: https://cdn.elifesciences.org/articles/58659/elife-58659-fig1-data1-v2.xlsx
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Many pancreatic progenitor-expressed lncRNAs are cytoplasmically enriched and translated

Although most functional roles described for lncRNAs to date have been predominantly nuclear (Marchese et al., 2017), multiple recent studies have shown that many lncRNAs are cytosolic and translated into sometimes biologically active microproteins (reviewed in Makarewich and Olson, 2017). To further characterize the above-identified dynamically regulated lncRNAs, we analyzed their subcellular localization and translation potential using fractionation RNA-seq and Ribo-seq across multiple hESC clones independently differentiated into PP2 stage pancreatic progenitors (Figure 2A). Of all lncRNAs expressed in two replicate differentiations into PP2 cells, we classified 21% (n = 347) as localized to the nucleus, whereas a larger number (n = 563; 34%) primarily resided in the cytosol (Figure 2—figure supplement 1A and Figure 2—source data 1A). This subcellular distribution of pancreatic lncRNAs is in agreement with previous lncRNA localization studies by us and others (Cabili et al., 2015; Clark et al., 2012; Sun et al., 2015; van Heesch et al., 2014). LncRNAs enriched in the cytosol were expressed at higher levels than nucleus-localized lncRNAs, with expression levels similar to canonical protein-coding mRNAs (Figure 2—figure supplement 1B). Intriguingly, almost half (49.4%) of all cytosol-enriched lncRNAs (278 out of 563) displayed dynamic expression regulation during the differentiation of hESCs to pancreatic progenitors, raising the possibility that many lncRNAs with putative developmental functions do not act in the nucleus, but instead in the cytosol where they may be translated.

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Cytosolic lncRNAs contain translated small open reading frames.

(A) Overview of experimental strategy for subcellular fractionation and Ribo-seq-based identification of translated small open reading frames (sORFs) from lncRNAs expressed in PP2 cells. Replicates from six independent differentiations to PP2 stage each for total (polyA) RNA-seq and Ribo-seq experiments, and two biological replicates for the subcellular fractionation were analyzed. The histogram on the far right depicts the size distribution of the sORF-encoded small peptides as number of amino acids (aa). The pie chart summarizes the percentages of constitutively and dynamically expressed sORF-encoding lncRNAs during pancreatic differentiation of CyT49 hESCs. (**B–E**) Left: Bar graphs showing nuclear and cytosolic expression (in RPKM) of lncRNAs *RP11-834C11.4* (B), *LINC00261* (C), *MIR7-3HG* (D), and *LHFPL3-AS2* (E). Data are shown as mean, with individual data points represented by dots (n = 2 biological replicates). Right: Subcellular fractionation RNA-seq, Ribo-seq, and P-site tracks (ribosomal P-sites inferred from ribosome footprints on ribosome-protected RNA) for loci of the depicted lncRNAs. Identified highest stringency sORFs (ORF in 6/6 replicates) are shown in red. For *LINC00261*, visually identified sORFs 1 and 2 are also shown. Heatmaps in the top right visualize the relative expression of the shown lncRNAs during pancreatic differentiation (means of two biological replicates per stage), on a minimum (white)/maximum (dark blue) scale. (F) In vivo translation reporter assays testing whether sORFs computationally defined in (A) give rise to translation products in HEK293T cells when fused in-frame to a GFP reporter. Left: Schematic of the constructs (gray: *PGK* promoter, black: lncRNA sequence 5’ to sORF to be tested, red: sORF, green: GFP ORF). Right: Representative DIC and GFP images of HEK293T cells transiently transfected with the indicated reporter constructs. Scale bars = 50 µm. See also Figure 2—figure supplement 1 and Figure 2—source data 1.

Figure 2—source data 1 RNA-seq after subcellular fractionation and Ribo-seq in PP2 cells. (A) Subcellular fractionation of PP2 stage cells (RPKM). (B) Ribo-seq/mRNA-seq contaminant filtering statistics, read size distribution, and Pearson correlation coefficients of all sequenced Ribo-seq and polyA RNA-seq libraries. (C) All ORFs detected by RiboTaper, including lncRNA sORFs. (D) lncRNA sORFs detected by RiboTaper and conservation statistics (PhyloCSF scores). (E) Translational efficiency calculations.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig2-data1-v2.xlsx
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To investigate the translation potential of these cytosolic lncRNAs, we used Ribo-seq, through which we obtained exceptionally deep and high quality translatome coverage across six replicate differentiations (Figure 2—figure supplement 1C and Figure 2—source data 1B). As nearly 90% of the sequenced ribosomal footprints exhibited clear 3-nucleotide codon movement characteristic of translation (Figure 2—figure supplement 1D–F), these data have strong predictive value for the computational detection of non-canonical ORFs, such as upstream ORFs (uORFs) in the 5' leader sequences of mRNAs and sORFs in genes annotated as lncRNAs (Figure 2—source data 1C). Requiring stringent reproducibility criteria (the exact ORF needed to be detected by RiboTaper (Calviello et al., 2016) in at least four out of six replicates), we identified a total of 625 new sORFs in lncRNAs with a median length of 47 amino acids (aa) (Figure 2—source data 1D). The majority of detected sORFs (76%; n = 477/625) is currently not present in the sORFs.org database (Olexiouk et al., 2016). The translated sORFs are located within 285 cytosolically localized lncRNAs (25.3% of all expressed lncRNAs) (Figure 2—figure supplement 1B), which are expressed at higher levels than untranslated lncRNAs (Figure 2—figure supplement 1G) and exhibit translational efficiencies similar to mRNAs (Figure 2—figure supplement 1H and Figure 2—source data 1E). Of note, almost none of the newly identified sORFs are highly conserved across species, as judged by their low PhyloCSF scores (Lin et al., 2011; Figure 2—source data 1D).

Using approaches similar to ours, non-canonical sORFs have previously been characterized in multiple immortalized human cell lines (Bazzini et al., 2014; Calviello et al., 2016; Chen et al., 2020; Ji et al., 2015; Martinez et al., 2020; Prensner et al., 2020; Raj et al., 2016) and human tissues (van Heesch et al., 2019). However, to our knowledge, our data constitute the first comprehensive set of non-canonical human ORFs generated from a non-transformed human cell model of development, providing a valuable resource for future functional studies.

Translated lncRNAs in pancreatic progenitors produce microproteins with distinct subcellular localizations

Having established that many stage-specific pancreatic lncRNAs are translated, we next sought to validate their translation potential through independent experimental approaches, additionally investigating the production of the predicted microproteins at the protein level. To this end, we first performed coupled in vitro transcription:translation assays on endogenous and complete transcript isoforms of four of the most highly translated lncRNAs (LINC00261, RP11-834C11.4, LHFPL3-AS2, and MIR7-3HG; Figure 2—figure supplement 1I; expression and ORF information in Figure 2B–E). Second, we generated a series of in vivo translation reporter constructs to assess the subcellular localization of microproteins translated from each of ten sORFs derived from the same four lncRNAs. Transient expression of individual constructs carrying in-frame GFP fusions in HEK293T cells produced GFP signal for all ten assayed microproteins, which was abolished upon introduction of a frameshift within the sORF or a stop codon following the sORF sequence (Figure 2F and Figure 2—figure supplement 1J–L). To rule out a possible localization bias induced by the GFP fusion, we also expressed a FLAG-tag fusion peptide (RP11-834C11.4 sORF-1xFLAG), which revealed a cytoplasmic localization identical to the one observed for the GFP construct (Figure 2—figure supplement 1J). While most sORF-GFP fusion products were ubiquitously distributed throughout transfected cells, LINC00261 sORF4-GFP specifically localized to mitochondria (Figure 2—figure supplement 1K), and LINC00261 sORF7-GFP exhibited a perinuclear accumulation pattern reminiscent of aggresomes (Figure 2—figure supplement 1L). Taken together, our results validate the translation potential of sORFs encoded by pancreatic progenitor-expressed lncRNAs and show that, upon ectopic expression, these translation events result in the production of microproteins with different subcellular localizations.

Deletion phenotypes of translated lncRNAs during hESC pancreatic differentiation

To identify potential functional roles of translated lncRNAs during pancreas development, we selected ten candidates for CRISPR/Cas9-based genome editing in hESCs through excision of the lncRNA promoter or entire lncRNA locus (Figure 3A,B). These ten lncRNAs were prioritized based on (i) high expression and endodermal tissue-specificity, (ii) dynamic regulation during pancreas development, (iii) abundant translation of sORFs, and (iv) proximity to TFs with known roles in endoderm and pancreas development. For seven of the selected lncRNAs, translation was highly abundant and reproducibly detected across Ribo-seq replicates: LINC00617 (also known as TUNAR; Lin et al., 2014), GATA6-AS1 (also known as GATA6-AS; Neumann et al., 2018), LINC00261, RP11-834C11.4, SOX9-AS1, MIR7-3HG, and LHFPL3-AS2. Although for two additional lncRNAs the translation potential could not be determined, they were nonetheless included because of a previously reported requirement for definitive endoderm formation (DIGIT, also known as GSC-DT) (Daneshvar et al., 2016) and genomic localization adjacent to the definitive endoderm TF LHX1 (RP11-445F12.1, also known as LHX1-DT). Lastly, LINC00479 was chosen as a non-translated control with expression dynamics and a subcellular localization similar to LINC00261. Of note, for each of the ten selected lncRNAs, we generated at least two independent hESC knockout (KO) clones and used different combinations of single guide RNAs where possible (Figure 3—source data 1A).

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A small-scale CRISPR loss-of-function screen for dynamically expressed and translated lncRNAs during pancreatic differentiation.

(A) qRT-PCR analysis of candidate lncRNAs during pancreatic differentiation of H1 hESCs relative to the ES stage. Data are shown as mean ± S.E.M. (mean of n = 2–6 independent differentiations per stage; from H1 hESCs). Individual data points are represented by dots. See also Figure 3—source data 2. (B) CRISPR-based lncRNA knockout (KO) strategy in H1 hESCs and subsequent phenotypic characterization. (C) Immunofluorescence staining for OCT4 and SOX17 in DE from control (ctrl) and KO cells for the indicated lncRNAs (representative images, n ≥ 3 independent differentiations; at least two KO clones were analyzed). (D) qRT-PCR analysis of DE lineage markers in DE from control and lncRNA KO (-/-) cells. TF genes in cis to the lncRNA locus are highlighted in red. Data are shown as mean ± S.E.M. (n = 3–16 replicates from independent differentiations and different KO clones). Individual data points are represented by dots. NS, p-value>0.05; t-test. See also Figure 3—source data 3. (E) Flow cytometry analysis at DE stage for SOX17 in control and KO (-/-) cells for indicated lncRNAs. The line demarks isotype control. Percentage of cells expressing SOX17 is indicated (representative experiment, n ≥ 3 independent differentiations from at least two KO clones). (F) Immunofluorescence staining for FOXA2 or GATA6 in DE from control and *LINC00261*, *GATA6-AS1*, and *DIGIT* KO cells. (G) Immunofluorescence staining for insulin (INS) in endocrine cell stage (EC) from control and KO hESCs for the indicated lncRNAs (representative images, n ≥ 3 independent differentiations from at least two KO clones). Boxed areas (dashed boxes) are shown in higher magnification. (H) qRT-PCR analysis of *INS* in EC stage cultures from control and lncRNA KO (-/-) hESCs. Data are shown as mean ± S.E.M. (n ≥ 4 replicates from independent differentiations of at least two KO clones). Individual data points are represented by dots. NS, p-value>0.05; t-test. See also Figure 3—source data 4 (I) Flow cytometry analysis at EC stage for INS in control and KO (-/-) cells for indicated lncRNAs. The line demarks isotype control. Percentage of cells expressing insulin is indicated (representative experiment, n ≥ 3 independent differentiations each from at least two KO clones). Scale bars = 100 µm. See also Figure 3—figure supplement 1 and Figure 3—source data 1–4.

Figure 3—source data 1 Differentially expressed genes after lncRNA deletion. (A) Coordinates of CRISPR deletions. (B) Differentially expressed genes in RP11-445F12.1 knockout at definitive endoderm stage. (C) Differentially expressed genes in GATA6-AS1 knockout at definitive endoderm stage. (D) Differentially expressed genes in LINC00261 knockout at definitive endoderm stage. (E) Differentially expressed genes in LINC00617 knockout at PP2 stage. (F) Differentially expressed genes in GATA6-AS1 knockout at PP2 stage. (G) Differentially expressed genes in LINC00479 knockout at PP2 stage. (H) Differentially expressed genes in RP11-834C11.4 knockout at PP2 stage. (I) Differentially expressed genes in SOX9-AS1 knockout at PP2 stage. (J) Differentially expressed genes in MIR7-3HG knockout at PP2 stage. (K) Differentially expressed genes in LHFPL3-AS2 knockout at PP2 stage.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig3-data1-v2.xlsx
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Figure 3—source data 2 Source data used for the qRT-PCR quantification of gene expression presented in Figure 3A.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig3-data2-v2.xlsx
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Figure 3—source data 3 Source data used for the qRT-PCR quantification of gene expression presented in Figure 3D.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig3-data3-v2.xlsx
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Figure 3—source data 4 Source data used for the qRT-PCR quantification of INS expression presented in Figure 3H.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig3-data4-v2.xlsx
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We next differentiated each of the lncRNA KO hESC lines stepwise toward the pancreatic endocrine cell stage, conducting up to 16 replicate differentiations for each KO clone. Because LINC00617, RP11-445F12.1, DIGIT, GATA6-AS1, LINC00479, and LINC00261 were first expressed at, or before, the definitive endoderm stage (Figure 3A), we determined whether KO hESCs for these lncRNAs exhibited defects in definitive endoderm formation. Despite efficient lncRNA depletion (Figure 3—figure supplement 1A,B), neither quantification of definitive endoderm marker gene expression by qRT-PCR, nor immunofluorescence staining or flow cytometric analysis of the definitive endoderm marker SOX17 showed differences indicative of impaired endoderm formation in lncRNA KO lines (Figure 3C–E). Importantly, expression of TFs located in the direct vicinity of these lncRNAs, including GSC (DIGIT), LHX1 (RP11-445F12.1), GATA6 (GATA6-AS1), and FOXA2 (LINC00261), was unaffected by the lncRNA KO (Figure 3F, Figure 3—figure supplement 1C, Figure 3—source data 1B–D), arguing against cis-regulation by these lncRNAs. These findings are in contrast to prior reports that have shown a requirement for LINC00261 and DIGIT in definitive endoderm formation and the regulation of neighboring TFs FOXA2 and GSC, respectively (Amaral et al., 2018; Daneshvar et al., 2016; Jiang et al., 2015; Swarr et al., 2019).

Next, we further differentiated control and KO lines for eight out of ten lncRNAs toward the endocrine cell stage, excluding DIGIT and RP11-445F12.1 because they are not expressed after the definitive endoderm stage (Figure 3A). In KO hESC lines of seven out of these eight lncRNAs, we observed no effect on pancreatic progenitor cell formation or gene expression, with the exception of a handful of dysregulated genes in LHFPL3-AS2 and RP11-834C11.4 KO cells (Figure 3—figure supplement 1C and Figure 3—source data 1E–K). Furthermore, deletion of seven out of the eight lncRNAs did not impair endocrine cell formation, as determined by quantification of insulin⁺ cells and insulin mRNA levels (Figure 3G–I). Similar to the RNA expression results obtained at the definitive endoderm stage, deletion of none of the lncRNAs close to pancreatic TFs (e.g. GATA6-AS1 and SOX9-AS1) altered the expression of these TFs, once more arguing against cis-regulation of these TFs by the neighboring lncRNA (Figure 3—figure supplement 1C). Thus, nine out of ten endoderm- and pancreatic progenitor-enriched lncRNAs functionally investigated here appear to be nonessential for induction of the pancreatic fate and formation of insulin⁺ cells. Furthermore, these lncRNAs do not appear to control the transcript levels of proximal TFs.

LINC00261 knockout impairs endocrine cell development

The exception was the endoderm-specific lncRNA LINC00261, which is highly expressed and translated in pancreatic progenitors (Figure 4—figure supplement 1A and Figure 2C). While deletion of LINC00261 caused no discernable phenotype in definitive endoderm (Figure 3C–F and Figure 3—figure supplement 1C), we observed a significant 30–50% reduction in the number of insulin⁺ cells at the endocrine cell stage (Figure 4A,B). This reduction in insulin⁺ cell numbers was consistent across four separately derived LINC00261 KO hESC lines, each independently differentiated to endocrine cell stage 5–8 times. In agreement with reduced insulin⁺ cell numbers, insulin content and insulin mRNA levels were also reduced in LINC00261 KO endocrine stage cultures (Figure 4C,D). Analysis of insulin median fluorescence intensities by flow cytometry further showed no reduction in insulin levels per cell in one LINC00261 KO clone and a mild reduction in the three other clones (Figure 4E), indicating that LINC00261 predominately regulates endocrine cell differentiation rather than maintenance of insulin production in beta cells.

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*LINC00261* deletion impedes pancreatic endocrine cell differentiation.

(A) Flow cytometry analysis at endocrine cell stage (EC) for insulin (INS) in control (ctrl) and *LINC000261^-/-* H1 hESCs. Top panel: Schematic of the *LINC00261* locus. The dashed box represents the genomic deletion. Middle panel: The line demarks isotype control. Percentage of cells expressing INS is indicated (representative experiment, n = 4 deletion clones generated with independent sgRNAs). Bottom panel: Bar graph showing percentages of INS-positive cells. Data are shown as mean ± S.D. (n = 5 (clone 1), n = 6 (clone 2), n = 8 (clone 3), n = 5 (clone 4) independent differentiations). Individual data points are represented by dots. (B) Immunofluorescence staining for INS in EC stage cultures from control and *LINC000261*^-/- hESCs (representative images, number of differentiations see A). Boxed areas (dashed boxes) are shown in higher magnification. (C) ELISA for INS in EC stage cultures from control and *LINC00261*^-/- hESCs. Data are shown as mean ± S.D. (n = 3 (clone 1), n = 2 (clone 2), n = 14 (clone 3), n = 13 (clone 4) independent differentiations). Individual data points are represented by dots. (D) qRT-PCR analysis of *INS* in EC stage cultures from control and *LINC00261*^-/- hESCs. Data are shown as mean ± S.E.M. (n = 8 (clone 1), n = 4 (clone 2), n = 10 (clone 3), n = 3 (clone 4) independent differentiations). Individual data points are represented by dots. (E) Quantification of median fluorescence intensity after INS staining of control and *LINC00261*^-/- EC stage cultures. Data are shown as mean ± S.D. (n = 5 (clone 1), n = 5 (clone 2), n = 4 (clone 3), n = 4 (clone 4) independent differentiations). iso, isotype control. Individual data points are represented by dots. (F) Volcano plot displaying gene expression changes in control versus *LINC00261*^-/- PP2 cells (n = 6 independent differentiations from all four deletion clones). Differentially expressed genes are shown in red (DESeq2;>2 fold change (FC), adjusted p-value<0.01) and blue (>2 fold change, adjusted p-value≥0.01 and≤0.05). Thresholds are represented by vertical and horizontal dashed lines. *FOXA2* in cis to *LINC00261* is shown in gray (gray dots represent genes with ≤ 2 fold change and/or adjusted p-value>0.05). (G) Circos plot visualizing the chromosomal locations of the 108 genes differentially expressed (DESeq2;>2 fold change (FC), adjusted p-value<0.01) in *LINC00261*^-/- compared to control PP2 cells, relative to *LINC00261* on chromosome 20. No chromosome was over- or underrepresented (Fisher test, p-value>0.05 for all chromosomes). (H) Top panel: Schematic of the *LINC00261* locus, with the location of its sORFs (1 to 7) marked by vertical red bars. Bottom panel: Flow cytometric quantification of INS-positive cells in control and *LINC00261*-sORF-frameshift (FS) at the EC stage. Data are shown as mean ± S.D. (n = 4–7 independent differentiations per clone). (I) ELISA for INS in EC stage cultures from control and *LINC00261*-sORF-FS hESCs. Data are shown as mean ± S.D. (n = 3–7 independent differentiations per clone). (J) Volcano plot displaying gene expression changes in control versus *LINC00261*-sORF3-FS PP2 cells. No gene was differentially expressed (DESeq2;>2 fold change, adjusted p-value<0.01; indicated by dashed horizontal and vertical lines; n = 2 independent differentiations). *LINC00261* is shown in gray, the bar graph insert displays *LINC00261* RPKM values in control and *LINC00261*-sORF3-FS PP2 cells. (K) *LINC00261* half-life measurements in HEK293T cells transduced with lentivirus expressing either wild type (WT) *LINC00261* or ΔATG^sORF1-7 *LINC00261* (mutant in which the ATG start codons of sORFs 1–7 were changed to non-start codons). HEK293T were treated with the transcription inhibitor actinomycin D and RNA isolated at 0, 2, 4, 6, 8, and 9 hr post actinomycin D addition. *LINC00261* expression was analyzed by qRT-PCR relative to the *TBP* gene. Data are shown as mean ± S.E.M. (n = 3 biological replicates for each assay time point). *, p-value<0.05; **, p-value<0.01; ***, p-value<0.001; ****, p-value<0.0001; NS, p-value>0.05; t-test. Scale bars = 100 µm. See also Figure 4—figure supplement 1 and Figure 4—source data 1–3.

Figure 4—source data 1 Characterization of LINC00261 knockout and LINC00261-sORF3-frameshift PP2 cells. (A) Differentially expressed genes in LINC00261 knockout PP2 stage cells. (B) Sequences of LINC00261 wild type and frameshift mutants. (C) Differentially expressed genes in LINC00261-sORF3-frameshift PP2 stage cells.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig4-data1-v2.xlsx
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Figure 4—source data 2 List of oligonucleotides and synthetic gene fragments used in this study. (A) sgRNA oligonucleotides used for cloning into PX458/Px459. (B) Genotyping and sequencing primers for KO validation. (C) qRT-PCR primers. (D) Cloning primers (translation reporter constructs and lentiviral LINC00261 overexpression plasmids). (E) Synthetic gene fragments. (F) Custom LINC00261 Stellaris RNA FISH probe set.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig4-data2-v2.xlsx
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Figure 4—source data 3 Source data used for the insulin measurements presented in Figure 4.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig4-data3-v2.xlsx
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To determine the molecular effects of LINC00261 deletion, we performed RNA-seq in pancreatic progenitors derived from LINC00261 KO and control hESCs. Similar to the absence of cis-regulatory functions observed in the other lncRNA KOs, we found no evidence for cis-regulation of FOXA2 by LINC00261 (Figure 4F and Figure 4—figure supplement 1C). However, we observed downregulation of the TFs MAFB and PAX4 (Figure 4F, Figure 4—figure supplement 1B, Figure 4—source data 1A), which are important regulators of beta cell differentiation (Artner et al., 2007; Sosa-Pineda et al., 1997). Of note, genes differentially expressed in LINC00261 KO cells mapped to all chromosomes and showed no enrichment for chromosome 20 where LINC00261 resides (Figure 4G). These results suggest a trans- rather than cis-regulatory function for LINC00261, consistent with its predominantly cytosolic localization, translation, and diffuse distribution within the nucleus (Figure 2C and Figure 4—figure supplement 1D). Trans-regulatory roles of LINC00261 have also been observed in previous studies (Aguet et al., 2019; Shi et al., 2019; Wang et al., 2019; Wang et al., 2017; Yan et al., 2019). This potential trans functionality prompted us to further investigate whether LINC00261's coding or noncoding features are essential for endocrine cell differentiation.

One-by-one disruption of LINC00261's sORFs does not impact endocrine cell differentiation

We established that LINC00261 harbors multiple distinct and highly translated sORFs, which raises the possibility that the translation of these sORFs is functionally important for endocrine cell differentiation. To systematically discriminate LINC00261's coding and noncoding roles, we individually mutated its seven most highly translated sORFs independently in hESCs, leaving the lncRNA sequence, and hence any noncoding function coupled to RNA sequence or structure, grossly intact. Each of these hESC lines either carried a homozygous frameshift mutation near the microprotein's N-terminus (for sORFs 1–6) or a full sORF deletion (sORF7; Figure 4—source data 1B). After verifying that CRISPR editing of the LINC00261 locus did not impact LINC00261 transcript levels (Figure 4—figure supplement 1E), we quantified (i) insulin mRNA levels, (ii) insulin⁺ cells, and (iii) total insulin content in endocrine cell stage cultures. We observed no difference between sORF loss-of-function and control hESC lines for most of these endpoints (Figure 4H,I and Figure 4—figure supplement 1E), although we noticed that the number of insulin⁺ cells, but not the amount of insulin produced, was reduced in one of the two sORF4 and sORF7 KO clones. Transcriptome analysis of pancreatic progenitors with frameshifts in sORF3 (the most highly translated LINC00261-sORF; Figure 2C and Figure 2—source data 1D) revealed no differentially expressed genes between LINC00261-sORF3 frameshift and control cells (Figure 4J and Figure 4—source data 1C), contrasting observations in LINC00261 RNA KO pancreatic progenitors (Figure 4F and Figure 4—source data 1A). These results indicate that there is not one dominant LINC00261 sORF that is required for endocrine cell formation, suggesting a functional role of the LINC00261 transcript and not the individual sORFs mutated here. However, it is possible that the different sORFs, or the microproteins translated from these sORFs, are functionally redundant and capable of phenotypic rescue.

It has been suggested that ribosome association can control lncRNA transcript levels by inducing nonsense-mediated decay (NMD) (Carlevaro-Fita et al., 2016; Tani et al., 2013). Therefore, we determined whether the presence of multiple sORFs could regulate LINC00261 stability. To this end, we simultaneously mutated start codons of all seven sORFs (ΔATG^sORF1-7 LINC00261) and expressed either wild type or ΔATG^sORF1-7 LINC00261 ectopically in HEK293T cells, where LINC00261 is normally not expressed. LINC00261 half-life measurements upon transcriptional inhibition with actinomycin D revealed no difference in LINC00261 levels between wild type and ΔATG^sORF1-7 LINC00261 (Figure 4K), suggesting that the translation of the seven sORFs does not reduce LINC00261 transcript stability.

In sum, through the systematic, one-by-one removal of sORFs within a highly translated lncRNA with functional importance for pancreatic endocrine cell formation, we found no evidence to implicate the individual sORFs, or the microproteins they produce, in endocrine cell development. Although LINC00261's sORFs may share functional redundancy or have developmental roles that do not affect the production of insulin⁺ cells, our findings strongly suggest that by themselves, these sORFs are not functionally required for endocrine cell formation.

Discussion

Limited cis-regulatory consequences of lncRNA deletion

In this study we globally characterized molecular features of lncRNAs expressed during progression of hESCs toward the pancreatic lineage, including their subcellular localization and potential to be translated. We performed a phenotypic CRISPR loss-of-function screen, focusing on ten developmentally regulated, highly expressed, and highly translated lncRNAs proximal to TFs known to regulate pancreas development. The first important observation from this screen is that we found no evidence to implicate the lncRNAs LINC00261, DIGIT, GATA6-AS1, SOX9-AS1, and RP11-445F12.1 in the cis-regulation of their neighboring TFs FOXA2, GSC, GATA6, SOX9, and LHX, respectively, despite tight transcriptional coregulation of the lncRNA-TF pairs.

Contrasting our findings, a number of studies have reported cis-regulation of FOXA2 by LINC00261 (Amaral et al., 2018; Jiang et al., 2015; Swarr et al., 2019). However, several lines of evidence strongly support the conclusion that FOXA2 is not regulated by LINC00261 in our experimental system. First, we examined FOXA2 mRNA expression in LINC000261^-/- cells at both the definitive endoderm and pancreatic progenitor cell stages. Second, we analyzed FOXA2 expression using two independent methods, namely qRT-PCR and RNA-seq. Third, immunofluorescence staining in definitive endoderm revealed no difference in FOXA2 protein expression between control and LINC00261^-/- cells.

While different cellular contexts and species could explain the discrepancy between our findings and the ones by Amaral et al., 2018 and Swarr et al., 2019, Jiang et al., 2015 reported FOXA2 regulation by LINC00261 in hESC-derived definitive endoderm. One important difference between our study and the study by Jiang et al. is that we employed CRISPR-Cas9-mediated deletion, whereas Jiang et al. used shRNA-mediated knockdown to inactivate LINC00261. It is possible that lncRNA deletion triggers compensatory mechanisms that are not activated after shRNA-mediated knockdown. For coding genes, mutant mRNA degradation has been shown to trigger genetic compensation (El-Brolosy et al., 2019). Another difference between our study and the one by Jiang et al. is that our differentiation protocol was more efficient in generating definitive endoderm. It is conceivable that the stability of the cell fate and identity of neighboring cells could influence how LINC00261 loss-of-function affects gene regulation.

Translation of short, non-canonical ORFs in lncRNAs: regulatory, microprotein-producing, or just tolerated?

Although lncRNAs are now appreciated as a novel and abundant source of sORF-encoded biologically active microproteins (Makarewich and Olson, 2017), it remains largely unknown which translation events lead to the production of microproteins, which solely have regulatory potential, or which have no functional roles, but are not negatively selected against. The cytosolic localization and translation of many RNAs classified as lncRNAs provides a strong rationale for considering both, coding and noncoding functions.

In this study, we identified the translated lncRNA LINC00261 as a novel regulator of pancreatic endocrine cell differentiation, as evidenced by a severe reduction in insulin⁺ cell numbers upon LINC00261 deletion. We show that LINC00261 transcripts are highly abundant in pancreatic progenitors and, albeit present in the nucleus, are predominantly localized to the cytoplasm. Here, they frequently associate with ribosomes which leads to the translation of multiple independent sORFs. We show that the sORFs are capable of producing microproteins with distinct subcellular localizations upon expression in vitro. In contrast to LINC00261 deletion, individual frameshift mutations in each of LINC00261's sORFs did not impair endocrine cell development, suggesting that the requirement of LINC00261 for endocrine cell development can be uncoupled from the translation of its multiple sORFs. However, this does not exclude the possibility that these sORFs or the microproteins they produce could possess functions that become relevant under specific environmental, developmental, or disease conditions not examined in this study.

We found that mutating all translated LINC00261 sORFs simultaneously, thereby likely reducing LINC00261's ability to bind ribosomes, did not affect LINC00261 transcript levels in HEK293T cells. This indicates that, in contrast to reports suggesting that translated sORFs can regulate RNA stability by promoting nonsense-mediated RNA decay (Carlevaro-Fita et al., 2016; Tani et al., 2013), the high translation levels and multiple sORFs of LINC00261 are unlikely to be part of a LINC00261 decay pathway. It would have been interesting to determine how concurrent mutation of all sORFs in LINC00261 affects pancreatic cell differentiation. However, given the size of the LINC00261 locus and the many sORFs, such an approach comes with technical challenges and significant caveats.

LINC00261 - a potential trans regulator of endocrine cell differentiation?

Several lines of evidence suggest that LINC00261 regulates endocrine cell differentiation in trans: (i) LINC00261 transcripts show a diffuse distribution in multiple subcellular compartments, (ii) genes differentially expressed in LINC00261 KO cells are randomly distributed throughout the genome, (iii) expression of the nearby TF FOXA2 is not affected by LINC00261 deletion. Such a trans regulatory mechanism for LINC00261 is supported by a recent study from the GTEx Consortium, where LINC00261 is highlighted as one of a few lncRNAs that forms a potential trans regulatory hotspot through genetic interactions that influence the expression of multiple distant genes (Aguet et al., 2019). Consistent with its preferential cytosolic localization, and further supporting the notion of a trans regulatory mechanism, LINC00261 has been suggested to regulate gene expression through non-nuclear mechanisms, e.g. by preventing nuclear translocation of β-catenin (Wang et al., 2017) or by acting as a miRNA sponge (Shi et al., 2019; Wang et al., 2019; Yan et al., 2019). Although our observations and current literature strongly hint to a function in trans independent of the produced microproteins, the exact mechanism by which LINC00261 regulates gene expression in pancreatic progenitors remains to be determined.

Limitations and future directions

In this study, we have characterized the role of translated lncRNAs, and in particular LINC00261, in a hESC differentiation system that mimics pancreas development. However, there are several potential limitations that need to be considered when interpreting the results. First, a small subset of analyses in this study was based on low numbers of replicate differentiations, in particular the cytosolic versus nuclear fractionation RNA-seq experiments, where only two replicate differentiations into pancreatic progenitor cells were analyzed. Second, although we provide evidence that LINC00261 can produce microproteins using Ribo-seq, which is further supported by in vitro translation assays and overexpression of LINC00261 constructs with different in-frame tags, we provide no protein-level evidence for the endogenous production and stability of LINC00261's microproteins in this differentiation system or in human pancreas development in vivo. Moreover, due to its highly specific expression pattern, LINC00261 has not been previously detected by sORF analyses in other cell types (Bazzini et al., 2014; Calviello et al., 2016; Chen et al., 2020; Ji et al., 2015; Martinez et al., 2020; Prensner et al., 2020; Raj et al., 2016; van Heesch et al., 2019). Even though we show microprotein production in vitro, it is possible that the act of translation has a key regulatory role rather than the protein products of LINC00261’s sORFs. Lastly, LINC00261's microproteins and sORFs may have redundant functions, which could explain why deletion of individual sORFs produces no apparent phenotype. Thus, despite limited sequence similarity and stark differences in translation rates between the identified translated sORFs in LINC00261, we cannot rule out that different microproteins produced by LINC00261 compensate when one sORF is deleted. Future studies of LINC00261's precise mechanisms of action could be aimed at further dissecting the potential regulatory features of sORF translation and possibility of redundancy between sORFs.

Conclusions

In summary, we here present a rigorous, in-depth characterization of dynamically regulated and translated lncRNAs in a disease-relevant cell model of human developmental progression. Our combination of ultra-high-coverage RNA- and Ribo-seq, in vitro protein-level validation of microprotein production and localization, and the systematic, one-by-one deletion of all individual microproteins encoded by a single translated lncRNA, not only provides a detailed resource of translated 'non-canonical' sORFs and their microproteins in pancreatic development, but also serves as a blueprint for the systematic functional interrogation of translated lncRNAs.

Materials and methods

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Homo sapiens)	LINC00617; TUNAR	Ensembl 87	ENSG00000250366
Gene (Homo sapiens)	RP11-445F12.1; LHX-DT	Ensembl 87	ENSG00000250366
Gene (Homo sapiens)	DIGIT; GSC-DT	HGNC and NCBI RefSeq	HGNC:53074; NCBI RefSeq 108868751
Gene (Homo sapiens)	GATA6-AS1; GATA6-AS	Ensembl 87	ENSG00000277268
Gene (Homo sapiens)	LINC00479	Ensembl 87	ENSG00000236384
Gene (Homo sapiens)	LINC00261; DEANR1; ALIEN; onco-lncRNA-17; lnc-FOXA2-2	Ensembl 87	ENSG00000236384
Gene (Homo sapiens)	RP11-834C11.4	Ensembl 87	ENSG00000250742
Gene (Homo sapiens)	SOX9-AS1	Ensembl 87	ENSG00000234899
Gene (Homo sapiens)	MIR7-3HG	Ensembl 87	ENSG00000176840
Gene (Homo sapiens)	LHFPL3-AS2	Ensembl 87	ENSG00000225329
Strain, strain background (Escherichia coli)	Stbl3	ThermoFisher Scientific	Cat# C737303	Chemically competent cells
Strain, strain background (Escherichia coli)	DH5α	New England Biolabs	Cat# C2987I	Chemically competent cells
Cell line (Homo sapiens)	H1 (embryonic stem cells)	WiCell Research Institute	NIHhESC-10–0043, RRID:CVCL_9771
Cell line (Homo sapiens)	HEK293T (embryonic kidney)	ATCC	Cat# CRL-3216, RRID:CVCL_0063
Antibody	anti-human OCT-4A (Rabbit monoclonal)	Cell Signaling Technology	Cat# 2890, RRID:AB_2167725	IF (1:1000)
Antibody	anti-human SOX17 (Goat polyclonal)	R and D Systems	Cat# AF1924, RRID:AB_355060	IF (1:250)
Antibody	anti-human FOXA2 (Goat polyclonal)	Santa Cruz Biotechnology	Cat# sc-6554, RRID:AB_2262810	IF (1:250)
Antibody	anti-human GATA6 (Goat polyclonal)	Santa Cruz Biotechnology	Cat# sc-9055, RRID:AB_2108768	IF (1:50)
Antibody	anti-human Insulin (Guinea pig polyclonal)	Dako	Cat# A0564, RRID:AB_10013624	IF (1:1000)
Antibody	Alexa Fluor 488 AffiniPure anti-Goat IgG (Donkey polyclonal)	Jackson ImmunoResearch Labs	Cat# 706-545-148, RRID:AB_2340472	IF (1:1000)
Antibody	Alexa Fluor 488 AffiniPure anti-Rabbit IgG (Donkey polyclonal)	Jackson ImmunoResearch Labs	Cat# 711-545-152, RRID:AB_2313584	IF (1:1000)
Antibody	Cy3 AffiniPure anti-Goat IgG (Donkey polyclonal)	Jackson ImmunoResearch Labs	Cat# 705-165-147, RRID:AB_2307351	IF (1:1000)
Antibody	anti-human Insulin-PE (Rabbit monoclonal)	Cell Signaling Technology	Cat# 8508S, RRID:AB_11179076	Flow cytometry (1:50)
Antibody	anti-human SOX17-PE (Mouse monoclonal)	BD Biosciences	Cat# 561591, RRID:AB_10717121	Flow cytometry (5 ul per test)
Antibody	IgG-PE (Rabbit monoclonal)	Cell Signaling Technology	Cat# 5742S, RRID:AB_10694219	Flow cytometry isotype control antibody (1:50)
Antibody	IgG1, κ antibody (Mouse monoclonal)	BD Biosciences	Cat# 556650, RRID:AB_396514	Flow cytometry isotype control antibody (1:50)
Recombinant DNA reagent	pSpCas9(BB)−2A-Puro (Px459; V2.0)	Feng Zhang	RRID:Addgene_62988	Cas9 from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA
Recombinant DNA reagent	pSpCas9(BB)−2 A-GFP (PX458)	Feng Zhang	RRID:Addgene_48138	Cas9 from S. pyogenes with 2A-EGFP, and cloning backbone for sgRNA
Recombinant DNA reagent	pCMVR8.74	Didier Trono	RRID:Addgene_22036	2nd generation lentiviral packaging plasmid
Recombinant DNA reagent	pMD2.G	Didier Trono	RRID:Addgene_12259	VSV-G envelope expressing plasmid
Recombinant DNA reagent	pENTR/D-TOPO-LINC00261	Kurian et al., 2015 (PMID:25739401)		Leo Kurian (University of Cologne)
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-GFP.WPRE	Didier Trono	RRID:Addgene_12252	3rd generation lentiviral backbone
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of wild type LINC00261
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-ΔATG^sORF1-7 LINC00261.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of ΔATG^sORF1-7 LINC00261
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF1-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF1-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF2-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF2-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF3-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF3-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF4-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF4-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF5-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF5-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF6-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF6-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF7-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF7-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF3-FS-GFP.WPRE	This paper		LINC00261-sORF3-frameshift-GFP control plasmid
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF2-STOP-GFP.WPRE	This paper		LINC00261-sORF2-STOP-GFP control plasmid
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-RP11-834C11.4-sORF-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of RP11-834C11.4-sORF-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-RP11-834C11.4-sORF-FLAG.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of RP11-834C11.4-sORF-FLAG fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-MIR7-3HG-sORF-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of MIR7-3HG-sORF-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-RP11-LHFPL3-AS2-sORF-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LHFPL3-AS2-sORF-GFP fusion protein
Peptide, recombinant protein	Activin A	R and D Systems	Cat# 338-AC/CF
Peptide, recombinant protein	Wnt3a	R and D Systems	Cat# 1324-WN-010
Peptide, recombinant protein	KGF/FGF7	R and D Systems	Cat# 251 KG
Peptide, recombinant protein	Noggin	R and D Systems	Cat# 3344 NG-050
Commercial assay or kit	RNeasy Mini Kit	QIAGEN	Cat# 15596018
Commercial assay or kit	RNA Clean and Concentrator−25	Zymo Research	Cat# R1018
Commercial assay or kit	Paris Kit	Thermo Fisher Scientific	Cat# AM1921
Commercial assay or kit	RNase-Free DNase Set (50)	QIAGEN	Cat# 79254
Commercial assay or kit	TURBO DNA-free Kit	Thermo Fisher Scientific	Cat# AM1907
Commercial assay or kit	TruSeq Ribo Profile (Mammalian) Library Prep Kit	Illumina	Cat# RPYSC12116
Commercial assay or kit	TruSeq Stranded mRNA Library Prep	Illumina	Cat# 20020594
Commercial assay or kit	TruSeq Stranded Total RNA Library Prep Gold	Illumina	Cat# 20020599
Commercial assay or kit	KAPA mRNA HyperPrep Kit	Roche	Cat# KK8581
Commercial assay or kit	High Sensitivity D1000 ScreenTape	Agilent Technologies	Cat# 5067–5584
Commercial assay or kit	RNA ScreenTape	Agilent Technologies	Cat# 5067–5576
Commercial assay or kit	RNA ScreenTape Sample Buffer	Agilent Technologies	Cat# 5067–5577
Commercial assay or kit	RNA ScreenTape Ladder	Agilent Technologies	Cat# 5067–5578
Commercial assay or kit	Qubit ssDNA assay kit	Thermo Fisher Scientific	Cat# Q10212
Commercial assay or kit	KOD Xtreme DNA Hotstart Polymerase	Millipore	Cat# 71975
Commercial assay or kit	GoTaq Green Master Mix	Promega	Cat# M7123
Commercial assay or kit	TOPO TA Cloning Kit	Thermo Fisher Scientific	Cat# K452001
Commercial assay or kit	Monarch Plasmid Miniprep Kit	NEB	Cat# T1010L
Commercial assay or kit	MinElute PCR Purification Kit	QIAGEN	Cat# 28006
Commercial assay or kit	iScript cDNA Synthesis Kit	Bio-Rad	Cat# 1708890
Commercial assay or kit	iQ SYBR Green Supermix	Bio-Rad	Cat# 1708880
Commercial assay or kit	Human Stem Cell Nucleofector Kit 2	Lonza	Cat# VPH-5022
Commercial assay or kit	XtremeGene 9 DNA Transfection Reagent	Sigma-Aldrich	Cat# 06365779001
Commercial assay or kit	Cytofix/Cytoperm W/Golgi Stop Kit	BD Biosciences	Cat# 554715
Commercial assay or kit	Insulin ELISA Jumbo	Alpco	Cat# 80-INSHU-E10.1
Commercial assay or kit	Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	Cat# 23227
Commercial assay or kit	TnT Coupled Wheat Germ Extract System	Promega	Cat# L4130
Chemical compound, drug	Penicillin-Streptomycin	Thermo Fisher Scientific	Cat# 15140122
Chemical compound, drug	Puromycin dihydrochloride	Sigma-Aldrich	Cat# P8833
Chemical compound, drug	ALK5 Inhibitor II	Enzo Life Sciences	Cat# ALX-270–445
Chemical compound, drug	Retinoic Acid	Sigma-Aldrich	Cat# R2625
Chemical compound, drug	Ascorbic Acid	Sigma-Aldrich	Cat# A4403-100MG
Chemical compound, drug	LDN-193189	Stemgent	Cat# 04–0074
Chemical compound, drug	SANT-1	Sigma-Aldrich	Cat# S4572
Chemical compound, drug	TPB	Calbiochem	Cat# 565740
Chemical compound, drug	TGFβ R1 kinase inhibitor IV	EMD Biosciences	Cat# 616454
Chemical compound, drug	KAAD-Cyclopamine	Toronto Research Chemicals	Cat# K171000
Chemical compound, drug	TTNPB	Sigma-Aldrich	Cat# T3757
Chemical compound, drug	Cycloheximide	Sigma-Aldrich	Cat# C7698
Chemical compound, drug	Actinomycin D	Sigma-Aldrich	Cat# A9415
Chemical compound, drug	Polyethylenimine (PEI)	Polysciences	Cat# 23966–1
Chemical compound, drug	Hoechst 33342, Trihydrochloride, Trihydrate	Thermo Fisher Scientific	Cat# H3570
Chemical compound, drug	MitoSOX Red	Thermo Fisher Scientific	Cat# M36008
Chemical compound, drug	D-(+)-Glucose Solution, 45%	Sigma-Aldrich	Cat# G8769
Chemical compound, drug	Sodium Bicarbonate	Sigma-Aldrich	Cat# NC0564699
Chemical compound, drug	ROCK Inhibitor Y-27632	STEMCELL Technologies	Cat# 72305
Software, algorithm	Flowjo-v10	FlowJo, LLC	RRID:SCR_008520	http://www.flowjo.com/download-newest-version/
Software, algorithm	STAR 2.5.3a	Dobin et al., 2013	RRID:SCR_015899	https://github.com/alexdobin/STAR
Software, algorithm	Bowtie 1.1.1	Langmead et al., 2009	RRID:SCR_005476	http://bowtie-bio.sourceforge.net/index.shtml
Software, algorithm	Cufflinks 2.2.1	Trapnell et al., 2010	RRID:SCR_014597	https://github.com/cole-trapnell-lab/cufflinks
Software, algorithm	HTSeq 0.6.1	Anders et al., 2015	RRID:SCR_005514	https://htseq.readthedocs.io/en/master/install.html
Software, algorithm	DEseq2 1.10.1	Love et al., 2014	RRID:SCR_015687	https://www.bioconductor.org/packages/devel/bioc/html/DESeq2.html
Software, algorithm	RiboTaper	Calviello et al., 2016	RRID:SCR_018880	https://ohlerlab.mdc-berlin.de/software/RiboTaper_126/
Software, algorithm	R 3.5.0		RRID:SCR_001905	https://cran.r-project.org/
Software, algorithm	SAMtools 1.3	Li et al., 2009	RRID:SCR_002105	https://github.com/samtools/samtools
Software, algorithm	BEDTools 2.17.0	Quinlan and Hall, 2010	RRID:SCR_006646	https://bedtools.readthedocs.io/en/latest/content/installation.html
Software, algorithm	HOMER 4.10	Heinz et al., 2010	RRID:SCR_010881	http://homer.ucsd.edu/homer/download.html
Software, algorithm	GREAT 3.0.0	McLean et al., 2010	RRID:SCR_005807	http://great.stanford.edu/public/html/
Software, algorithm	Adobe Illustrator CS5	Adobe	RRID:SCR_010279
Software, algorithm	Adobe Photoshop CS5	Adobe	RRID:SCR_014199
Software, algorithm	GraphPad Prism v7.05	GraphPad Software, LLC	RRID:SCR_002798
Other	Novex 16% Tricine Protein Gel	Thermo Fisher Scientific	Cat# EC66955BOX
Other	Novex Tricine SDS Sample Buffer (2X)	Thermo Fisher Scientific	Cat# LC1676
Other	Immobilon-PSQ PVDF Membrane	Merck Millipore	Cat# ISEQ00010
Other	Stellaris RNA FISH Hybridisation Buffer	LGC Biosearch Technologies	Cat# SMF-HB1-10
Other	Stellaris RNA FISH Wash Buffer A	LGC Biosearch Technologies	Cat# SMF-WA1-60
Other	Stellaris RNA FISH Wash Buffer B	LGC Biosearch Technologies	Cat# SMF-WB1-20
Other	QuickExtract DNA Extraction Solution	Lucigen	Cat# QE09050
Other	Vectashield Antifade Mounting Medium	Vector Laboratories	Cat# H-1000
Other	FastDigest BpiI	Thermo Fisher Scientific	Cat# FD1014
Other	FastDigest BshTI	Thermo Fisher Scientific	Cat# FERFD1464
Other	FastDigest SalI	Thermo Fisher Scientific	Cat# FD0644
Other	TRIzol	Thermo Fisher Scientific	Cat# 15596018
Other	Matrigel	Corning	Cat# 356231
Other	GlutaMAX	Thermo Fisher Scientific	Cat# 35050061
Other	DPBS (without calcium and magnesium)	Corning	Cat# 45000–434
Other	mTeSR1 Complete Kit - GMP	STEMCELL Technologies	Cat# 85850
Other	RPMI 1640 Medium, HEPES	Thermo Fisher Scientific	Cat# 22400–089
Other	DMEM/F12 with L-Glutamine, HEPES	Corning	Cat# 45000–350
Other	Dulbecco’s Modified Eagle’s Medium	Corning	Cat# 45000–312
Other	HyClone Dulbecco’s Modified Eagles Medium	Thermo Fisher Scientific	Cat# SH30081.FS
Other	MCDB 131	Thermo Fisher Scientific	Cat# 10372–019
Other	Opti-MEM Reduced Serum Medium	Thermo Fisher Scientific	Cat# 31985062
Other	Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) (100X)	Thermo Fisher Scientific	Cat# 51500–056
Other	B-27 Supplement (50X)	Thermo Fisher Scientific	Cat# 17504044
Other	Bovine Albumin Fraction V (7.5%)	Thermo Fisher Scientific	Cat# 15260037
Other	Fetal Bovine Serum	Corning	Cat# 35011CV
Other	Donkey Serum	Gemini Bio-Products	Cat# 100-151/500
Other	Fatty Acid-Free BSA	Proliant Biologicals	Cat# 68700
Other	Accutase	eBioscience	Cat# 00-4555-56

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LncRNA expression and regulation during pancreatic differentiation.

Figure 1—source data 1

Cytosolic lncRNAs contain translated small open reading frames.

Figure 2—source data 1

A small-scale CRISPR loss-of-function screen for dynamically expressed and translated lncRNAs during pancreatic differentiation.

Figure 3—source data 1

Figure 3—source data 2

Figure 3—source data 3

Figure 3—source data 4

LINC00261 deletion impedes pancreatic endocrine cell differentiation.

Figure 4—source data 1

Figure 4—source data 2

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Bjoern Gaertner

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Sebastiaan van Heesch

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Valentin Schneider-Lunitz

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Jana Felicitas Schulz

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Franziska Witte

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Susanne Blachut

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Steven Nguyen

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Regina Wong

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Ileana Matta

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Norbert Hübner

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Maike Sander

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For correspondence

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