The vast majority of mammalian DNA replicates in homologous regions of chromosome pairs in a highly synchronous manner (Mukhopadhyay et al., 2014; Dileep and Gilbert, 2018; Rivera-Mulia et al., 2018). However, genetic disruption of non-protein coding ASAR (‘ASynchronous replication and Autosomal RNA’) loci causes a delay in replication timing on individual human chromosomes in cis, resulting in highly asynchronous replication between pairs of autosomes (Donley et al., 2015; Heskett et al., 2020; Heskett et al., 2022; Stoffregen et al., 2011). There are eight genetically validated ASARs that are located on human chromosomes 1, 6, 8, 9, and 15 (Donley et al., 2015; Heskett et al., 2020; Heskett et al., 2022; Stoffregen et al., 2011). All of the known ASAR lncRNAs share several distinctive characteristics, including: (1) contiguously transcribed regions of >180 kb, (2) epigenetically regulated allelic expression imbalance (AEI; including mono-allelic expression); (3) variable epigenetic replication timing (VERT; including asynchronous replication timing); (4) high density of LINE1 (L1) sequences (>30%); and (5) retention of the RNA within their parent chromosome territory. Recently, we used these notable characteristics to identify 68 ASAR candidates on every human autosome (Heskett et al., 2020; Heskett et al., 2022).

In addition to genetic disruption assays, ectopic integration of ASAR transgenes into mouse chromosomes has been used as a second functional assay to help define the critical sequences within ASAR lncRNAs that control chromosome-wide replication timing (Donley et al., 2013; Platt et al., 2018). For example, ASAR6 lncRNA expressed from transgenes, ranging in size from an ~180 kb BAC transgene to PCR products (as small as ~3 kb) containing the critical sequences, remain associated with the chromosome territories where they are transcribed (Donley et al., 2013; Platt et al., 2018). These same transgenes cause delayed replication timing (DRT) and delayed mitotic condensation (DMC) of entire chromosomes in cis (Donley et al., 2013; Platt et al., 2018). Furthermore, ASAR6 lncRNA was found to control chromosome-wide replication timing using oligonucleotide-mediated RNA degradation of ASAR6 lncRNA that was expressed from a BAC transgene integrated into mouse chromosome 3 (Platt et al., 2018). These findings suggest that ASARs represent ubiquitous, essential, cis-acting elements that control mammalian chromosome replication timing via expression of chromosome associated RNAs. In this report, we identify RNA binding proteins (RBPs) that are critical for ASAR lncRNA localization and function.

Chromosome-associated long non-coding RNAs are known to be important for numerous aspects of chromosome dynamics in diverse eukaryotes from yeast to mammals (Ding et al., 2019; Faber and Vo, 2022; Nozawa and Gilbert, 2019; Creamer et al., 2021; Menon and Meller, 2015). The mammalian nucleus is rich in heterogenous nuclear RNA (hnRNA), which represents >95% of the total RNA Polymerase II output (Nozawa and Gilbert, 2019; St Laurent et al., 2012). The vast majority of hnRNA is poorly defined, has been referred to as the ‘Dark Matter’ of the genome (St Laurent et al., 2012), and includes spliced and unspliced intronic sequences, circular RNAs (Kristensen et al., 2019), very long intergenic non-coding RNAs (vlincRNAs; St Laurent et al., 2016), as well as poorly defined RNAs that include chromatin associated RNAs (caRNAs; Nozawa et al., 2017) and repeat-rich RNAs (repRNAs; Frank and Rippe, 2020) that can be detected by C0T-1 DNA (which is comprised primarily of LINE and SINE repetitive sequences; Creamer et al., 2021; Hall et al., 2014; Kolpa et al., 2022). Some of these nuclear RNAs comprise abundant species, yet lack clearly defined functions, while others are thought to be the by-products of other nuclear processes and have been collectively called ‘RNA debris’ (Nozawa and Gilbert, 2019). The relationship between ASAR lncRNAs and these other chromosome-associated RNAs remains poorly defined.

Previous studies have suggested that long nascent transcripts that are detected by C0T-1 DNA play a dynamic structural role that promotes the open architecture of active chromosome territories (Creamer et al., 2021; Hall et al., 2014). The RNA species detected by C0T-1 DNA are predominantly L1 sequences, and these L1 RNAs remain associated with the chromosome territories where they are transcribed (Hall et al., 2014). A link between C0T-1 RNA and ASAR lncRNAs is suggested by the observation that ASAR lncRNAs contain a high L1 content and remain associated with their parent chromosome territories (Donley et al., 2015; Heskett et al., 2020; Heskett et al., 2022; Stoffregen et al., 2011; Platt et al., 2018). In addition, deletion and ectopic integration assays demonstrated that the critical sequences for controlling chromosome-wide replication timing within ASAR6 and ASAR15 map to the antisense strand of L1 sequences located within the ASAR6 and ASAR15 lncRNAs (Platt et al., 2018). In this report we found that human C0T-1 DNA can detect the chromosome territory localization of ASAR6 lncRNA that is expressed from a BAC transgene integrated into a mouse chromosome, indicating that at least some of the RNA species detected by C0T-1 DNA represents ASAR lncRNA.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of abundant RBPs that contribute to multiple aspects of nucleic acid metabolism. Many of the proteins that are in hnRNP complexes share general features, but differ in domain composition and functional properties (reviewed in Geuens et al., 2016). The functions of hnRNPs vary according to their subcellular localization. Most of the hnRNPs possess conventional nuclear localization signals and are predominantly present in the nucleus during steady state, where they function in various RNA metabolic processes including transcription, splicing, nuclear export, and 3’ end processing (Geuens et al., 2016; Domanski et al., 2022), and more recently have been implicated in DNA replication (Connolly et al., 2022). A direct connection between chromosome territory associated C0T-1 RNA and hnRNPs was established by the observation that siRNA mediated depletion, or forced expression of dominant interfering mutants, of HNRNPU results in dissociation of C0T-1 RNA from chromosome territories (Creamer et al., 2021; Hall et al., 2014; Kolpa et al., 2022). In addition, recent studies have proposed that abundant nuclear proteins such as HNRNPU nonspecifically interact with ‘RNA debris’ that creates a dynamic nuclear mesh that regulates interphase chromatin structure (Nozawa and Gilbert, 2019; Nozawa et al., 2017). Here, we used publicly available enhanced Cross Link Immuno-Precipitation (eCLIP) data from ENCODE (Moore et al., 2020) to identify RBPs, including HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, and HNRNPUL1, that interact with the known ASAR lncRNAs. We also found that an ~7 kb region within the ~185 kb ASAR6-141 lncRNA has a surprisingly high density of interacting RBPs, and that genetic deletion of this ~7 kb ‘RBP domain’ from the endogenous ASAR6-141 locus results in a delayed replication timing phenotype that is comparable to deletion of the entire ~185 kb ASAR6-141 transcribed region located on human chromosome 6. In addition, ectopic integration of transgenes, expressing the ~7 kb RBP domain, into autosomes or into the inactive X chromosome results in retention of the RNA within the chromosome territory of the integrated chromosomes, and results in DRT/DMC, and chromosome structure instability in cis. Taken together, these results indicate that the ~7 kb RBP domain represents a critical region within ASAR6-141 that controls replication timing of human chromosome 6.

Because ASARs were identified as cis-acting elements that control chromosome-wide replication timing, we tested if ASAR associated RBPs also control replication timing. We found that shRNA mediated depletion of 9 different ASAR-associated RBPs (HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, HLTF, KHSRP, or UCHL5) dramatically altered the normally synchronous replication timing program of all autosome pairs, recapitulating the effect of individual ASAR knockouts on a genome-wide scale. These results demonstrate the role that ASARs play during the temporal order of genome-wide replication, and suggests that ASAR lncRNAs serve as essential RNA scaffolds for the assembly of multiple hnRNP/RBP complexes that function to maintain the structural integrity of each mammalian chromosome.

All of the eight genetically validated ASARs are subject to AEI and express lncRNAs that remain associated with the chromosome territories where they are transcribed (Donley et al., 2015; Heskett et al., 2020; Heskett et al., 2022; Stoffregen et al., 2011). Examples of mono-allelic expression and chromosome territory localization of ASAR6-141 lncRNA are shown in Figure 1A and B, where ASAR6-141 lncRNA is localized within one of the chromosome 6 territories in two different cell types, male HTD114 cells and female GM12878 cells, respectively. We note that the size of the RNA hybridization signals detected by all ASAR probes are variable, ranging in size from large clouds that occupy entire chromosome territories, with similarity in appearance to XIST RNA ‘clouds’ (Figure 1B), to relatively small sites of hybridization that remain tightly associated with the expressed alleles (Donley et al., 2015; Heskett et al., 2020; Heskett et al., 2022; Stoffregen et al., 2011) .

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Figure 1—source data 1

The location of significant peaks of eCLIP reads that map within ASAR and XIST genes.

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Figure 1—source data 2

eCLIP reads for 120 RBPs in 10 kb windows within ASARs.

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Figure 1—source data 3

Fosmids, BACs, and primers.

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We first sought to identify RBPs that interact with the known ASAR lncRNAs. Using publicly available eCLIP data for 150 RBPs in K562 and HepG2 cells (ENCODE; Moore et al., 2020; Minks and Brown, 2009), we identified RBP interactions within 4 ASARs (ASAR1-187, ASAR6-141, ASAR8-2.7, and ASAR9-23). Utilizing the eCLIP peaks previously identified by ENCODE (Moore et al., 2020) we found significant RBP binding peaks for 99 different RBPs that map within the four ASAR lncRNAs (Figure 1—source data 1). We also utilized a ‘region-based’ method (Van Nostrand et al., 2016) to identify significant enrichments of eCLiP reads against matched controls in 10 kb windows across the four ASAR loci (Figure 1—source data 2). An example of this analysis is shown in Figure 1C, where ASAR6-141 displays significant RBP interactions for 35 different RBPs across the transcribed region. Given the contiguous expression (RNAseq from total RNA) across the ~185 kb ASAR6-141 locus in both K562 and HepG2 cell lines (Figure 1G) and the abundant eCLIP reads from 35 different RBPs in 10 kb windows across the ASAR6-141 lncRNA (Figure 1C and D; see Figure 1—source data 2), we were surprised to detect an ~7 kb region that contained virtually all of the significant eCLIP peaks in both cell lines (Figure 1G; and Figure 1—source data 1). The zoomed in view in Figure 1G shows alignment of 58 independent eCLIP peaks of RBP interactions within the ~7 kb RNA binding protein domain (RBPD). Figure 1E shows the 23 RBPs with the most abundant peaks of eCLIP reads within the ~7 kb RBPD. The eCLIP peaks associated with the RBPs chosen for shRNA knockdown experiments (see below) are highlighted in the zoomed in view in Figure 1G.

Disruption of the ~7kb RBPD within ASAR6-141 results in delayed replication

To determine if the ~7 kb RBPD within the ASAR6-141 lncRNA contains replication timing activity, we used CRISPR/Cas9-mediated engineering to delete the ~7 kb sequence from the endogenous locus on human chromosome 6. For this analysis, we designed single guide RNAs (sgRNAs) to unique sequences on either side of the ~7 kb RBPD (see Figure 1G; and Figure 1—figure supplement 1). We expressed these sgRNAs in combination with Cas9 in human HTD114 cells and screened clones for deletion of the ~7 kb RBPD (see Figure 1—figure supplement 1). We chose HTD114 cells for this analysis because they maintain mono-allelic expression and asynchronous replication of both imprinted and random monoallelic genes, they have a stable karyotype that does not change significantly following transfection, drug selection and sub-cloning, and knockouts of the eight known ASARs, including ASAR6-141, result in DRT/DMC and chromosome structure instability in cis (Donley et al., 2015; Heskett et al., 2020; Heskett et al., 2022; Stoffregen et al., 2011; Donley et al., 2013; Platt et al., 2018; Chang et al., 2007; Breger et al., 2005). Because ASAR6-141 expression is mono-allelic in the HTD114 cells (see Figure 1A; and Heskett et al., 2020), we isolated clones that had heterozygous or homozygous deletions of the ~7 kb RBPD. We determined which alleles were deleted based on retention of different base pairs of a heterozygous SNP located within the ~7 kb RBPD (see Figure 1—figure supplement 1 and Figure 1—source data 3).

To assay replication timing of homologous chromosome pairs we quantified DNA synthesis in mitotic cells using a BrdU terminal label assay (Figure 2A; and Smith et al., 2001; Smith and Thayer, 2012). In addition, the HTD114 cells contain a centromeric polymorphism on chromosome 6, which allows for an unambiguous distinction between the two chromosome 6 homologs (Stoffregen et al., 2011; Donley et al., 2013; Platt et al., 2018; also see Figure 2B). For simplicity, we refer to the chromosome 6 with the larger centromere as CHR6A and to the chromosome 6 with the smaller centromere as CHR6B. From our previous studies we knew that the chromosome 6 with the smaller centromere is linked to the expressed allele of ASAR6-141 ⁵. Figure 2B and C shows the replication timing analysis on a mitotic cell where the ~7 kb RBPD was deleted from the expressed allele (CHR6B). Note that CHR6B contains more BrdU incorporation than CHR6A, indicating later replication of CHR6B (Figure 2D). Quantification of the BrdU incorporation in CHR6B and CHR6A in multiple cells indicated that deletion of the ~7 kb RBPD from the expressed allele resulted in a significant delay in replication timing (Figure 2E). This is in contrast to cells prior to deletion, or in cells containing a deletion of the ~7 kb RBPD from the silent allele (CHR6A), where the BrdU incorporation is comparable between CHR6A and CHR6B (Figure 2E). In addition, an asynchronous replication pattern was also present in cells with homozygous deletions of the 7 kb RBPD, with CHR6B representing the later replicating allele (Figure 2E). Furthermore, we found that the delayed replication associated with deletion of the ~7 kb RBPD on CHR6B is comparable to the delayed replication associated with deletion of the entire ASAR6-141 locus from the same chromosome (i.e. CHR6B; Figure 2E; also see Heskett et al., 2020). These results indicate that the ~7 kb RBPD contains functional sequences for controlling replication timing, and further supports the conclusion that the expressed allele but not the silent allele controls replication timing of human chromosome 6 (Heskett et al., 2020).

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Depletion of RBPs disrupts the chromosome territory localization of ASAR lncRNAs

The results described above identified 99 RBPs expressed in two different cell lines that interact with four different ASAR lncRNAs (see Figure 1—source data 1). Next, to determine if the ASAR associated RBPs function in the chromosome territory localization of ASAR lncRNAs, we analyzed ASAR lncRNA localization in cells with shRNA depletion of 14 different RBPs. Figure 3A–J shows examples of antibody staining for HNRNPU, HNRNPUL1, HNRNPC, HNRNPM, and HLTF in K562 cells expressing either empty vector or vectors expressing shRNAs to individual RBPs. Quantitation of the immunofluorescence for each RBP in empty vector versus shRNA expressing cells, indicated a significant depletion of each RBP (Figure 3K; also see Figure 3—figure supplement 1 for western blots).

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Next, to determine if the nuclear localization of ASAR6-141 lncRNA was affected by RBP depletions, we used RNA FISH assays in K562 cells expressing shRNAs against the 14 RBPs. For this analysis, we used RNA FISH to detected ASAR6-141 lncRNA, and because K562 cells are female we also included a probe for XIST RNA as positive control for the RNA FISH. Figure 3L shows the expected RNA FISH patterns for both ASAR6-141 and XIST lncRNAs in cells expressing empty vector. In contrast, cells expressing shRNAs directed at HNRNPU, HNRNPUL1, HNRNPC, HNRNPM, HLTF, HNRNPA1, HNRNPL, KHSRP, or UCHL5 showed a nearly complete absence of the highly localized nuclear RNA hybridization signals for ASAR6-141 lncRNA, and the appearance of large punctate cytoplasmic hybridization signals (Figure 3M–U). We also noticed an overall increase in a diffuse ASAR6-141 lncRNA nuclear and cytoplasmic hybridization signals. In contrast, cells expressing shRNAs directed at PTBP1, PTBP2, or MATR3 showed only the typical nuclear localized hybridization signals for both ASAR6-141 and XIST lncRNAs (Figure 3V; and Figure 3—figure supplement 2A–D). In addition, localized nuclear XIST RNA hybridization signals were detected in all shRNA treated cells (Figure 3M–V and Figure 3—figure supplement 2A–D). However, we detected cytoplasmic foci of XIST RNA hybridization that colocalized with the ASAR6-141 lncRNA, and a significant decrease in the size of the nuclear XIST RNA hybridization signals (Figure 3W). These observations indicate that at least some of the XIST RNA was dissociated from the inactive X chromosome and colocalized with ASAR6-141 lncRNA in large cytoplasmic foci.

Ectopic integration of the ~7kb RBPD transgene into the inactive X chromosome

The results described above indicate that shRNA depletion of nine different RBPs resulted in dissociation of ASAR6-141 lncRNA from its parent chromosome, and resulted in a decreased nuclear XIST RNA hybridization signal with concomitant colocalization of both RNAs in cytoplasmic foci (see Figure 3), suggesting that ASAR6-141 and XIST lncRNAs are retained on their respective chromosome territories via similar mechanisms, which is supported by the large overlap in RBPs associated with both RNAs (Figure 1E and F; and Figure 1—source data 1). In addition, ectopic integration of Xist transgenes into autosomes has been an instrumental tool in characterization of Xist functions, including the ability to delay replication timing and induce gene silencing (Minks and Brown, 2009; Pintacuda et al., 2017; Lee et al., 1996). Therefore, we next sought to determine the consequences of ectopic integration of the ASAR6−141~7 kb RBPD transgene into the inactive X chromosome. The ectopic integration assays described above involved random integration of transgenes followed by an RNA FISH screen for clones expressing the ~7 kb RBPD, which included an RNA FISH probe for Xist RNA as positive control. Therefore, to identify transgene integrations into the inactive X chromosome, we simply screened an additional 50 clones for colocalization of RNA FISH hybridization signals for both Xist and the ~7 kb RBPD transgene. Figure 4A shows this RNA FISH assay on cells where the Xist and ~7 kb RBPD hybridization signals are colocalized. We note that the intensity of the Xist RNA FISH signal was enhanced in the region where the ~7 kb RBPD hybridization signal was detected (Figure 4B), suggesting that the presence of the ~7 kb RBPD RNA promoted preferential localization of Xist RNA in the same region of the X chromosome territory that contains the ~7 kb RBPD RNA.

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To confirm that the mouse X chromosome contains the transgene, we used DNA FISH using the ~7 kb RBPD DNA as probe in combination with an X chromosome paint probe on metaphase spreads. An example of this analysis is shown in Figure 2—figure supplement 1B–C, and indicates that the ~7 kb RBPD transgene is integrated into an X chromosome. During this analysis, we detected DMC on X chromosomes, and the X chromosomes with DMC contain the ~7 kb RBPD transgene (Figure 4C and D).

Next, to determine if the ~7 kb RBPD transgene alters replication timing of the inactive X chromosome, we visualized DNA synthesis in mitotic cells using our BrdU terminal label assay (see Figure 2A; and Smith et al., 2001; Smith and Thayer, 2012). Cells were exposed to BrdU for 5 hr, harvested for mitotic cells, and processed for BrdU incorporation and for FISH using the ~7 kb RBPD transgene plus an X chromosome paint probe to identify the integrated chromosome. Figure 4E–H shows an example of delayed replication and delayed mitotic condensation of the X chromosome in a mitotic cell containing the ~7 kb RBPD sense transgene integrated into the inactive X chromosome. We conclude that integration of the 7 kb RBPD transgene is sufficient to cause DRT/DMC on the inactive X chromosome.

Disruption of the chromosome associated localization of ASAR6 and ASAR1-187 lncRNAs

To determine if the chromosomal localization of additional ASAR RNAs was also affected by RBP knockdowns, we carried out RNA-DNA FISH for ASAR1-187 and ASAR6 lncRNAs. For this analysis, we used Fosmid probes to detect the ASAR RNAs plus BAC probes to detect the DNA near the ASAR loci located on chromosomes 1 and 6 (see Figure 1—source data 3). Figure 5A–C shows this RNA FISH assay on K562 cells expressing empty vector, and indicates that the RNA FISH signals for both ASARs are closely associated with the DNA FISH signals on their respective chromosomes, that is ASAR1-187 RNA shows close association with the chromosome 1 BAC DNA, and ASAR6 RNA shows close association with the chromosome 6 BAC DNA. In contrast, cells with shRNA depletion of HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, HLTF, KHSRP, or UCHL5 showed a dispersed punctate nuclear pattern for both ASAR RNAs (Figure 5D–K). In addition, large punctate cytoplasmic foci were detected, with the two ASAR RNA hybridization signals colocalized.

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Next, to determine if depletion of RBPs would result in loss of the ASAR RNA chromosome localization in a second cell type, we carried out RNA-DNA FISH assays on shRNA depleted HTD114 cells. We chose HTD114 cells because we previously used these cells to detect the chromosome territory localization of all eight ASAR RNAs and to measure the synchronous replication timing of five autosome pairs both before and after ASAR disruptions (Donley et al., 2015; Heskett et al., 2020; Heskett et al., 2022; Stoffregen et al., 2011; Donley et al., 2013; Breger et al., 2005; and see Figure 2). Figure 5L–N shows the RNA-DNA FISH assay in HTD114 cells expressing empty vector. We again note the tight association of the ASAR RNAs with the genomic loci where they are transcribed. In contrast, cells expressing shRNA against HNRNPU show loss of the chromosome associated RNA hybridization signals for both ASAR RNAs, and instead show diffuse nuclear and cytoplasmic hybridization, with the presence of large punctate cytoplasmic foci that hybridize to both ASAR RNA FISH probes (Figure 5O–Q). Similar results were obtained following shRNA depletion of HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPUL1, KHSRP, HLTF, or UCHL5 (Figure 5—figure supplement 1). In contrast, expression of shRNAs against SAFB, SAFB2, SAFB plus SAFB2, CTCF, or MATR3 did not alter the ASAR1-187 or ASAR6 RNA nuclear hybridization signals in HTD114 cells (Figure 3—figure supplement 2E–J).

Depletion of RBPs causes asynchronous replication on all autosome pairs

Next, to determine if ASAR associated RBPs function during the normally synchronous replication timing program that occurs on pairs of homologous chromosomes, we analyzed DNA synthesis in HTD114 cells depleted for the 9 RBPs implicated in ASAR RNA localization (HNRNPU, HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPUL1, KHSRP, HLTF, or UCHL5) and for five RBPs that showed no effect on ASAR RNA localization (MATR3, PTBP1, PTBP2, SAFB, or SAFB2). Using the BrdU terminal label assay (Smith et al., 2001; Smith and Thayer, 2012) in cells expressing the empty vector, we found that each autosome pair displays a stereotypical and synchronous BrdU incorporation pattern (Figure 6A). In contrast, following HNRNPU depletion, individual mitotic spreads displayed dramatic differential incorporation of BrdU into pairs of homologous chromosomes, examples of asynchronous replication timing between every pair of autosomes are shown in Figure 6B. Note that each chromosome pair contains dramatic differential BrdU incorporation into chromosome homologs, which represents considerable asynchronous replication timing. Figure 6C and D show examples for chromosome pairs 4 and 18 (also see Figure 6—figure supplement 1C). Quantification of the BrdU incorporation in chromosomes 1 and 6 (see Figure 6—figure supplement 1) in multiple cells depleted for HNRNPU, HNRNPUL1, HLTF, KHSRP, UCHL5, or HNRNPA1 indicated that both pairs of chromosomes display significant asynchrony (Figure 6E and F). In addition, the DRT/DMC phenotype was detected in ~30% of mitotic spreads following shRNA-mediated depletions (two examples are shown in Figure 6G and H; also see Figure 6—figure supplement 2). Furthermore, shRNA-mediated depletion of HNRNPC, HNRNPL and HNRNPM resulted in dramatic alterations in chromosome morphology that included asynchronous replication and abnormal mitotic condensation, including ~10% of mitotic spreads with one or more chromosomes that displayed the DRT/DMC phenotype (Figure 6—figure supplement 2).

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C0T-1 DNA hybridizes to ASAR6 RNA

Finally, to determine the relationship between the RNA species detected by C0T-1 DNA and ASAR RNAs in RNA FISH assays, we used human C0T-1 DNA as an RNA FISH probe on mouse cells expressing an ASAR6 BAC transgene (Donley et al., 2013). Because human C0T-1 DNA does not cross hybridize with mouse RNA or DNA in FISH assays (Hall et al., 2014), we were able to detect the chromosome territory localized expression of human ASAR6 RNA expressed from the transgene that is integrated into one of the mouse chromosome 3 homologs using human C0T-1 DNA as RNA FISH probe (Figure 7A and B), indicating that C0T-1 DNA detects ASAR6 RNA.

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We recently identified 68 ASAR candidates, and genetically validated five out of five as controlling replication timing of individual human autosomes, bringing the total of genetically validated ASARs to eight (Donley et al., 2015; Heskett et al., 2020; Heskett et al., 2022; Stoffregen et al., 2011). These eight ASARs (ASAR1-187, ASAR6, ASAR6-141, ASAR8-2.7, ASAR9-23, ASAR9-24, ASAR9-30, and ASAR15) display striking epigenetically controlled differential allelic expression and replication timing, and their RNAs remain localized to their parent chromosome territories (Donley et al., 2015; Heskett et al., 2020; Heskett et al., 2022; Stoffregen et al., 2011). In this report, we used publicly available RNA-protein interaction data from ENCODE to identify RBPs that interact with multiple ASAR lncRNAs. One unanticipated observation that came from this analysis was that an ~7 kb domain within the ~185 kb ASAR6-141 lncRNA contained virtually all of the significant peaks of RBP interactions in two different cell lines. Using genetic deletion and ectopic integration assays we found that this ~7 kb RBPD contains functional sequences for controlling replication timing of entire chromosomes in cis. We also found that shRNA-mediated depletion of 9 different ASAR-associated RBPs results in loss of the chromosome territory association of multiple ASAR RNAs. In addition, we found that depletion of HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, HLTF, KHSRP, or UCHL5 alters the normally synchronous replication timing that occurs between pairs of homologous chromosomes resulting in the DRT/DMC phenotype, recapitulating the effect of individual ASAR knockouts on a genome-wide scale.

Previous studies have suggested that C0T-1 RNA plays a dynamic structural role that promotes the open architecture of active chromosome territories (Creamer et al., 2021; Hall et al., 2014). C0T-1 RNA is predominantly L1 sequences, and these L1 RNAs remain associated with the chromosome territories where they are transcribed (Hall et al., 2014). Additional studies have proposed that repRNA is an important regulator of the dynamic organization of genomic loci into membrane-less subcompartments with distinct nuclear functions (reviewed in Frank and Rippe, 2020). Other studies have proposed that HNRNPU nonspecifically interacts with ‘RNA debris’ to create a dynamic nuclear mesh that regulates interphase chromatin structure (Nozawa and Gilbert, 2019; Nozawa et al., 2017). One important distinction that can be made between ASAR RNAs and these other hnRNAs (i.e. C0T-1 RNA, repRNA, caRNA, and ‘RNA debris’ Nozawa and Gilbert, 2019; Creamer et al., 2021; Nozawa et al., 2017; Frank and Rippe, 2020; Hall et al., 2014; Kolpa et al., 2022) is that ASARs have been defined genetically, and these other RNAs have not. Thus, we have used both loss-of-function and gain-of-function assays to define the roles that ASARs play in chromosome dynamics, which includes control of chromosome-wide replication timing and mitotic chromosome condensation (Donley et al., 2015; Heskett et al., 2020; Heskett et al., 2022; Stoffregen et al., 2011; Donley et al., 2013). We also found that C0T-1 DNA, when used as an RNA FISH probe, can detect ASAR6 RNA that is expressed and localized to an individual chromosome territory, indicating that at least some of the RNA FISH signal detected by C0T-1 DNA represents ASAR RNA. Given our recent discovery of ASAR RNAs expressed from every autosome, and encoded by ~3% of the human genome (Heskett et al., 2022), we propose that ASAR RNAs represent functional ‘chromosome associated RNA’ species that control chromosome dynamics within mammalian nuclei. Whether or not there are nuclear functions of other chromosome associated hnRNAs that are independent of ASARs will require genetic and/or functional validation.

Here we identified several hnRNPs (HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, and HNRNPUL1) as important interaction partners for the chromosome territory localization of multiple ASAR RNAs. In addition, we found that the SWI/SNF related transcription factor HLTF, which was recently shown to regulate replication fork reversal, prevent alternative mechanisms of stress-resistant DNA replication, and mediates cellular resistance to replication stress (Bai et al., 2020,) is required for the chromosome territory localization of multiple ASAR RNAs and for the synchronous replication of every autosome pair. Furthermore, we found that the KH domain (HNRNPK Homology domain) containing protein KHSRP, and the ubiquitin hydrolase UCHL5 also contribute to the chromosome territory localization of ASAR RNAs. We found that depletion of HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, HNRNPUL1, HLTF, KHSRP, or UCHL5 proteins resulted in a dramatic genome-wide disruption of the normally synchronous replication timing program on all autosome pairs. Taken together our results indicate that the association of ASAR RNAs with multiple hnRNPs in combination with other nuclear RBPs is an essential step in the regulation of the synchronous replication timing program on mammalian chromosomes.

While these studies have demonstrated a role for multiple RBPs in genome-wide chromosome replication timing, one limitation of these studies is that we only tested 14 out of the ~100 RBPs shown by eCLIP to associate with multiple ASAR RNAs. It is likely that additional insights into the network of RBP/ASAR interactions could be gained by extending our replication timing analyses to knockdowns of additional RBPs. Another limitation of these studies is that all of the RBPs implicated in control of replication timing are known to play various roles in other essential nucleic acid metabolic pathways, including transcription, splicing, mRNA export, and RNA degradation (Geuens et al., 2016; Domanski et al., 2022.) It is possible that knocking down these RBPs has an indirect effect on replication related to these other functions of the RBPs, rather than a direct effect on ASAR function. However, the RBP knockdowns that disrupt genome-wide synchronous replication also disrupt chromosome association of multiple ASAR RNAs. Our previous work demonstrated that the ability of an ectopically integrated ASAR6 transgene to cause delayed replication timing of a mouse chromosome required the ASAR6 RNA (Platt et al., 2018.) Hence, we expect that displacement of ASAR RNA from chromosome territories upon RBP knockdown is a primary cause of the genome-wide replication timing asynchrony, regardless of whether the displacement of ASAR RNA from the chromosome is a direct or indirect effect of RBP knockdown. A final limitation of these studies is that the precise mechanism by which ASARs and RBPs interact to control replication timing remains unknown. One intriguing possibility that is consistent with our data is that the ASAR RNAs serve as scaffolds for the assembly of extensive RNA-protein and protein-protein complexes that form a chromosome-tethered liquid-liquid phase that facilitates the recruitment of the requisite chromatin modifiers and DNA replication machinery. In support of this notion, HNRNPA1 and HNRNPC are components of the 40 S hnRNP particle, which forms a nuclear biomolecular condensate (Domanski et al., 2022). In addition, HNRNPU and HNRNPUL1 interact with each of the 40 S hnRNP particle subunits (Britton et al., 2014), suggesting the possibility of extensive protein-protein interactions. ASAR RNAs normally remain associated exclusively with the chromosome territory from which they are transcribed, and ASAR RNAs on different chromosomes remain separate. RBP knockdowns that disrupt synchronous replication timing also release ASAR RNAs from their chromosome territories, allowing colocalization of ASAR RNAs from different chromosomes (i.e. ASAR6 and ASAR1-187, see Figure 5O–Q), often in nuclear and/or cytoplasmic foci reminiscent of the mixing of liquid-liquid phases. Additional support for this model is the observation that Xist RNA participates in the assembly of a nuclear heteromeric condensate essential for gene silencing on the inactive X chromosome (Pandya-Jones et al., 2020). It is worth noting that ectopic integration of Xist transgenes has been a useful assay for characterization of Xist functions, including the ability to delay replication timing and induce gene silencing on autosomes (reviewed Minks and Brown, 2009). Thus, our observation that the ~7 kb RBPD RNA induces DRT/DMC on the inactive X chromosome, and associates with the Xist RNA cloud (Figure 4A and B), suggests that the ~7 kb RBPD RNA interferes with Xist RNA function within the heteromeric condensate. One intriguing observation, which is largely ignored by the X inactivation field, is that deletion of the Xist gene on either the active or inactive X chromosomes results in delayed replication timing of the X chromosome (Diaz-Perez et al., 2005; Diaz-Perez et al., 2006). Thus, loss of function mutations of either ASARs or Xist result in a similar chromosome-wide delayed replication timing phenotype. These parallels between ASAR and Xist mutation phenotypes suggest a shared mechanism for controlling replication timing in cis.

The formation of liquid-liquid phases via ASAR RNA-RBP interactions is speculative at this point and remains to be tested directly. A full understanding of the mechanisms by which ASARs control chromosome replication timing will require an extensive biochemical and biophysical characterization of the ASAR RNA-protein and protein-protein complexes and how they interact with the DNA replication machinery.

All data generated or analyzed during this study are included in the manuscript and supporting files; source data files have been provided for Figures 1, 2 and 3.

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Author details

1. Mathew Thayer

   Department of Chemical Physiology and Biochemistry,Oregon Health & Science University, Portland, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Contributed equally with

   Michael B Heskett

   For correspondence

   thayerm@ohsu.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6483-1661

2. Michael B Heskett

   1. Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, United States
   2. Stanford Cancer Institute, Stanford, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Mathew Thayer

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2089-3910

3. Leslie G Smith

   Department of Chemical Physiology and Biochemistry,Oregon Health & Science University, Portland, United States

   Contribution

   Data curation, Visualization

   Competing interests

   No competing interests declared

4. Paul T Spellman

   1. Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, United States
   2. Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, United States

   Present address

   Departments of Medicine and Human Genetics, University of California, Los Angeles, Los Angeles, United States

   Contribution

   Conceptualization, Data curation, Software, Funding acquisition

   Competing interests

   No competing interests declared

5. Phillip A Yates

   Department of Chemical Physiology and Biochemistry,Oregon Health & Science University, Portland, United States

   Contribution

   Conceptualization, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2016-9789

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