Fallatah et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00488-z(2023) 24:26

BMC Molecular and Cell
BiologyOpen Access

RESEARCH

Keratin 19 binds and regulates cytoplasmic
HNRNPK mRNA targets in triple-negative
breast cancer
Arwa Fallatah1,2, Dimitrios G. Anastasakis2, Amirhossein Manzourolajdad2,5, Pooja Sharma1, Xiantao Wang2,
Alexis Jacob2, Sarah Alsharif1, Ahmed Elgerbi1, Pierre A. Coulombe3,4, Markus Hafner2* and Byung Min Chung1*
Abstract
Background Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long noncoding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes
cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic
RNA ligands, and impact on post-transcriptional gene regulation remain uncharacterized.
Results Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK andsequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in thecytoplasm, resulting in reduced cell proliferation. We comprehensively mapped HNRNPK binding sites on mRNAs andshowed that, in the cytoplasm, K19-mediated HNRNPK-retention increases the abundance of target mRNAs boundto the 3’ untranslated region (3’UTR) at the expected cytidine-rich (C-rich) sequence elements. Furthermore, thesemRNAs protected by HNRNPK in the cytoplasm are typically involved in cancer progression and include the p53
signaling pathway that is dysregulated upon HNRNPK knockdown (HNRNPK KD) or K19 knockout (KRT19 KO).
Conclusions This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling thesubcellular localization of RNA-binding proteins to support pathways involved in cancer progression.
Keywords Keratin 19, HNRNPK, PAR-CLIP, Gene expression, Breast Cancer, p53, Proliferation, RNA binding protein,RNA-protein interaction*Correspondence:
Markus Hafnermarkus.hafner@nih.gov
Byung Min Chungchung@cua.edu
1
Department of Biology, The Catholic University of America, Washington,
DC, United States of America
2
RNA Molecular Biology Laboratory, National Institute of Arthritis andMusculoskeletal and Skin Diseases, Bethesda, MD, United States ofAmerica3
Department of Cell and Developmental Biology, University of Michigan
Medical School, Ann Arbor, MI, United States of America
4
Department of Dermatology, University of Michigan Medical School,
Ann Arbor, MI, United States of America
5
Present address: Department of Computer Science, Colgate University,
Hamilton, NY, United States of America

Background
Heterogeneous nuclear ribonucleoproteins (HNRNPs)
are a group of abundant nuclear RNA binding proteins(RBPs) consisting of more than 20 members from various RBP families that predominantly localize to the cell
nucleus [1]. One of the HNRNPs that promotes tumorigenesis is HNRNPK [2]. It comprises three K-homology(KH) RNA binding domains with a clear preference for
C-rich sequences [3], in addition to a less characterized
K interactive region (KI) that is thought to allow interaction with multiple other proteins [1]. HNRNPK plays
crucial roles in several biological processes, includingThis is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2023. Open Access This
article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution andreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative
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Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise
stated in a credit line to the data.

Fallatah et al. BMC Molecular and Cell Biology(2023) 24:26

development, axonal outgrowth, cell proliferation, andmigration [4–7]. In cancer, overexpression of HNRNPK
promotes tumor progression and correlates with poor
patient survival [8], likely by directly affecting the expression and activities of oncogenes and tumor suppressors
such as EIF4E [9], c-MYC [10], c-SRC [11] and MDM2[12]. As the list of oncogenes and tumor suppressors
regulated by HNRNPK is still growing, a comprehensive
perspective of HNRNPK-target genes and their regulatory mechanisms remain elusive. Considering that KH
domains typically only recognize a few nucleotides in
RNA [13], systems-wide methods are required for identifying HNRNPK-controlled posttranscriptional regulatory networks. While a few studies used crosslinking andimmunoprecipitation (CLIP)-type approaches to catalog
HNRNPK target mRNAs [14, 15], its genome-wide regulatory impact has not yet been determined.
While HNRNPK primarily resides in the nucleus [1],it impacts all steps of gene expression and a cytoplasmic pool of HNRNPK has been linked to metastasis andpoor prognosis in cancer [3, 16, 17]. HNRNPK contains
a nuclear shuttling domain [18], and posttranslational
modifications of this domain, such as phosphorylation
by ERK kinase [19] and methylation by PRMT1 [19]contribute to the regulation of the subcellular localization of HNRNPK. A previous study in skin cancer cells
showed that a cytoskeletal protein keratin 17 (K17) isrequired for the cytoplasmic localization of HNRNPK[4], demonstrating that keratin is a regulator of HNRNPK
localization.
The keratin family is composed of 54 genes, and theexpression of each keratin is tightly regulated in a tissue-,context-, and differentiation-specific manner. Among
keratins, keratin 19 (K19) is the smallest with a very shorttail domain. It is expressed in various simple and complex epithelial tissues and becomes upregulated in several
cancers where it is used as a diagnostic and prognostic
marker [20] Altered expression of K19 affects the growthof cancer cells in vitro and tumors in mice [20, 21], demonstrating its active role in cancer.
Inside the cell, keratins can interact with multiple proteins and regulate their localization. K19 has been shownto interact with various signaling molecules including β-catenin/RAC1[22], Egr1 [23], HER2 [24], andGSK3β [25] to regulate their subcellular localization. In
this study, we explored the possibility of an interaction
between K19 and HNRNPK and the indirect impact of
K19 on posttranscriptional gene regulation of HNRNPK
target mRNAs by changing the balance of nucleocytoplasmic HNRNPK localization. We found that the directinteraction between K19 and HNRNPK was required forcytoplasmic HNRNPK localization in the MDA-MB-231
breast cancer cells. By using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation

Page 2 of 13(PAR-CLIP) to identify mRNAs bound to HNRNPK
along with RNA-seq data, we identified genes whose
abundance are affected by K19. The top pathways ofgenes regulated by K19 and cytoplasmic HNRNPK wererelated to cancer signaling and included the p53 tumor
suppressor pathway. These findings reveal that K19 regulates HNRNPK functions in gene expression through its
physical interaction, ultimately affecting gene expression
in the mutant p53 signaling pathway. The oncogenic roleof K19 was confirmed by assessing cell proliferation in
the K19 context and finding that K19 is required for cellproliferation.

Results
K19 binds to HNRNPK and promotes its cytoplasmic
localization

We set out to study the effect of K19 on HNRNPK
localization and posttranscriptional gene regulatory
activity. We used MDA-MB-231 triple-negative breast
cancer (TNBC) cells with CRISPR-Cas9 mediated KRT19
knockout (KO) [26] and confirmed complete ablation ofK19 expression by Western blotting (Fig. S1A) and RNAsequencing (RNA-seq) (Table S1).
Keratins are an interaction platform for various proteins and a previous study showed that keratin closely
related to K19, K17, interacts with HNRNPK in skin cancer cells [4]. To test a potential interaction between K19
and HNRNPK, we performed proximity ligation assays(PLA). PLA using K19 and HNRNPK antibodies after
permeabilizing with a mild detergent (0.01% digitonin)
showed cytoplasmic fluorescence signals from parental
cells but not from KRT19 KO cells (Fig. 1A). PLA using
K19 and HNRNPK antibodies after permeabilizing withtriton also showed that K19 interacts with HNRNPK (Fig.
S1B). We further validated the K19-HNRNPK interaction
by co-immunoprecipitation (co-IP) of endogenous proteins and found that K19 and HNRNPK were detected
in IPs of HNRNPK and K19, respectively (Fig. 1B). Consistently, HNRNPK specifically co-immunoprecipitatedwith transiently expressed GFP-tagged K19 in HEK293
cells but not with the GFP control (Fig. 1C).
We then tested for a direct interaction between K19
filaments and HNRNPK in vitro by high-speed co-sedimentation. For this, filaments were preassembled using
purified K19 and its oligomerization partner keratin 8(K8). Next, recombinant HNRNPK was incubated withor without preassembled K8/K19 filaments. After highspeed centrifugation, sedimentation of recombinant
HNRNPK increased significantly in the presence of K8/
K19 filaments, whereas HNRNPK remained mostly in the
supernatant fraction without filaments (Fig. 1D), indicating a direct interaction between HNRNPK and K19
filaments.

Fallatah et al. BMC Molecular and Cell Biology(2023) 24:26

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Fig. 1 K19 binds to HNRNPK and promotes its cytoplasmic localization. (A) Proximity ligase assay showing K19 and HNRNPK colocalization in MDAMB-231 cells. Bar = 10 μm. (B) Co-IP of K19 and HNRNPK using IgG as a control. (C) Co-IP of K19 and HNRNPK using GFP and GFP-19 tagged transiently
expressing HEK293 cells. Full-length blots/gels are presented in Supplementary Fig. 7. (D) High-speed in vitro co-sedimentation assay following recombinant HNRNPK incubation with (+) or without (-) preassembled filaments of K19 and its obligate polymerization partner K8. Supernatant (S) and pellet (P)were separated, and immunoblotting was performed. (E) Biochemical subcellular fractionation showing decreased cytoplasmic HNRNPK in KRT19 knockout cells. PARP was used as a loading control for nuclear fraction while GAPDH was used as loading control for cytoplasmic fraction. Full-length blots/gels
are presented in Supplementary Fig. 8. (F) Quantification of cytoplasmic versus nuclear HNRNPK in MDA-MB-231 cells. (G) Immunofluorescence of cells(N = nucleus) permeabilized using mild detergent (bar = 10 μm). (H) Biochemical subcellular fractionation in both parental (P) and KRT19 KO cells showing
increased cytoplasmic HNRNPK in KRT19 KO cells upon overexpression K19. Full-length blots/gels are presented in Supplementary Fig. 9

Fallatah et al. BMC Molecular and Cell Biology(2023) 24:26

Keratin filaments regulate the subcellular localizationof several proteins [25, 27–29], and we hypothesizedthat K19 may influence the localization of the typically
predominantly nuclear HNRNPK. Therefore, we examined the cytoplasmic and nuclear levels of HNRNPK inparental and KRT19 KO cells. Subcellular fractionation
revealed a decrease in cytoplasmic HNRNPK level andcytoplasmic to the nuclear ratio of HNRNPK in KRT19
KO cells (Fig. 1E, F) without changes in overall HNRNPK
levels (Fig. S1A, C). Immunostaining of cytoplasmic
HNRNPK after mild permeabilization also showed ahigher HNRNPK signal from the cytoplasm of parental
cells compared to KRT19 KO cells (Fig. 1G). Finally, therescue of cytoplasmic HNRNPK levels by stable expression of GFP-K19 in KRT19 KO cells confirmed therequirement of K19 for cytoplasmic HNRNPK accumulation (Fig. 1H).
K19 regulates cytoplasmic HNRNPK targets

HNRNPK is expected to post-transcriptionally regulate
its RNA targets in the cytoplasm [16] and therefore we
examined whether loss of K19 and concomitant loss ofcytoplasmic HNRNPK resulted in changes in HNRNPK
target RNA levels. First, we comprehensively mapped the
RNA interactome of cytoplasmic HNRNPK and characterized its RNA recognition elements (RREs) using
4-thiouridine (4SU) PAR-CLIP in both parental and
KRT19 KO cells (Fig. S2) [30]. In these cells, RNA andproteins were crosslinked by UV, then subcellular fractionation of both parental and KRT19 KO cells was performed. HNRNPK from the cytoplasmic fraction was
immunoprecipitated, and the RNA covalently attachedin immunoprecipitated ribonucleoproteins (RNPs)
was partially digested and fluorescently labeled. SDSPAGE fluorescent imaging of the cytoplasmic HNRNPK
immunoprecipitated revealed two bands migrating at~ 60 kDa, corresponding to ribonucleoprotein complexes
containing HNRNPK (upper band) and its putative
spliced variant HNRNPJ (lower band) (Fig. 2A) [31, 32].
We recovered and sequenced cytoplasmic HNRNPKbound RNA fragments from MDA-MB-231 parental and
KRT19 KO cells and used PARalyzer to identify clustersof overlapping reads harboring the characteristic T-to-C
mutations indicating RNA-protein crosslinking events[33] (Tables S2, S3). Using HOMER, we found that our
PAR-CLIP binding sites were highly enriched for C/Urich sequences in both parental and KRT19 KO, consistent with the previously reported HNRNPK RRE [13, 34](Fig. 2B).
We next examined the role of K19 on the steady-state
abundance of HNRNPK targets and integrated RNAseq results from parental and KRT19 KO cells (Table S1)with our PAR-CLIP data. We found that the abundanceof cytoplasmic HNRNPK targets were reduced in KRT19

Page 4 of 13

KO cells, and the magnitude of this effect depended
on the overall strength of HNRNPK binding as definedby the number of cytoplasmic HNRNPK binding sites(Fig. 2C). In particular, levels of mRNAs with HNRNPKbinding sites at their 3’ untranslated region (UTR), coding, exon, and introns were decreased in KRT19 KO
cells (Fig. S3A-D). In contrast, mRNAs with HNRNPKbinding sites at the 5’ UTRs region were not affected in
KRT19 KO cells (Fig. S3E).
The cytoplasmic HNRNPK pool likely protects target mRNAs by increasing their steady-state abundance
through competition with other RBPs at 3’ UTR, analogous to the function of other RBPs [35]. Our findings
suggest that Keratin 19 plays a role in posttranscriptional
gene regulation by affecting subcellular localization of
HNRNPK, thereby altering the expression of HNRNPK
target mRNAs bound to HNRNPK via 3’ UTR.
To confirm the regulating role of K19 on HNRNPKdependent transcripts, we knocked down HNRNPK
using siRNAs in both parental and KRT19 KO cells (Fig.
S4). The abundance of HNRNPK targets in siHNRNPKtreated KRT19 KO cells was reduced compared to thosein siHNRNPK-treated parental cells (Fig. 2D), further
indicating that K19 regulates HNRNPK-dependenttranscripts. Additionally, cytoplasmic HNRNPK targets bound to 3’ untranslated region, coding, exon, andintrons were downregulated in KRT19 KO cells upon
knocking down HNRNPK, meaning that both K19 and
HNRNPK together are needed to maintain levels of cytoplasmic HNRNPK targets (Fig. S3F-J). Collectively, theseresults suggest that although K19 has little to no effect on
the mechanism of nucleic acid recognition by HNRNPK,
K19 indirectly affects the abundance of HNRNPK target
mRNAs by controlling its cytoplasmic accumulation.
K19 is required to maintain levels of cytoplasmic HNRNPK
target mRNAs

We then examined whether overexpressing cytoplasmic
HNRNPK can rescue the decreased levels of HNRNPK
targets occurring in the absence of K19. To specificallyoverexpress cytoplasmic HNRNPK, we created an expression construct with a deleted nuclear localization signal(HNRNPK ∆NLS), which indeed localized HNRNPK tothe cytoplasm (Fig. 3A-C). mCherry, which was used totag HNRNPK ∆NLS, did not affect the K19-mediated stabilization of cytoplasmic HNRNPK targets (Fig. S5). Integrating the list of cytoplasmic HNRNPK targets (Table
S2) with RNA-seq results from KRT19 KO cells overexpressing HNRNPK ∆NLS or vector (Table S4) showed nochange in levels of cytoplasmic HNRNPK targets (datanot shown). However, integrating the list of cytoplasmic
HNRNPK targets (Table S2) with RNA-seq results fromparental cells overexpressing HNRNPK ∆NLS or vector(Table S5) revealed a decrease in abundance of HNRNPKFallatah et al. BMC Molecular and Cell Biology(2023) 24:26

Page 5 of 13

Fig. 2 K19 regulates mRNAs directly bound to HNRNPK. (A) SDS-PAGE fluorescent imaging of the crosslinked cytoplasmic HNRNPK immunoprecipitation
revealed two bands migrating at ~ 60 kDa, corresponding to HNRNPK (upper band) and HNRNPJ (lower band), a putative spliced isoform of HNRNPK, in
both cell lines. (B) Sequence logo from cytoplasmic HNRNPK parental and KRT19 KO cells. (C) The empirical cumulative percentages plot of the change inmRNA levels in KRT19 KO compared to parental. The analysis compares HNRNPK targets (top 10%, Red, n = 733), (top 20%, Orange, n = 1488), (top 30%, Yellow, n = 2182), (top 40%, Green, n = 2930), and non-targets (Black, n = 3145). (D) The empirical cumulative percentages plot of the change in mRNA levels
in cells transiently depleted HNRNPK in both parental and KRT19 KO. The analysis compares HNRNPK targets (top 10%, Red, n = 870), (top 20%, Orange,n = 1761), (top 30%, Yellow, n = 2594), (top 40%, Green, n = 3480), and non-targets (Black, n = 1484). Targets (colored lines) binned by number of HNRNPK
binding sites and non-targets (black line) with minimal gene expression of 4 fragments per kilobase of exon per million mapped fragments ((FPKM) ≥ 4)
are showntarget mRNAs when HNRNPK ∆NLS was overexpressed(Fig. 3D). This decrease was independent of whether thetargets were binned by the overall number of HNRNPK
binding sites or by the number of binding sites only inthe target mRNA 3’ UTR (Fig. 3E). Altogether, these datasuggest that the ability of the HNRNPK to stabilize its
targets in the cytoplasm requires the presence of K19 as

well as the ability of HNRNPK to enter the nucleus andtransport targets from it.
K19 and HNRNPK co-regulate p53 signaling pathway

We next identified molecular pathways regulated by
HNRNPK that are dependent on K19 to find gene networks that may give insight into the apparent association

Fallatah et al. BMC Molecular and Cell Biology(2023) 24:26

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Fig. 3 Overexpressing cytoplasmic HNRNPK decreases levels of cytoplasmic HNRNPK targets. (A) Schematics of wildtype (WT) HNRNPK and cytoplasmic
mutant (ΔNLS). N-termini of WT and ΔNLS HNRNPK were tagged with mCherry (mCherry-WT and mCherry-ΔNLS, respectively). (B) Immunoblot fromlysates of KRT19 KO cells transfected with either mCherry, mCherry-WT, or mCherry-∆NLS using an antibody against mCherry. (C) KRT19 KO cells transiently
transfected with mCherry, mCherry-WT, or mCherry-∆NLS were immunostained with anti-RFP antibody. Nuclei are shown with DAPI (bar = 20 μm). The
empirical cumulative distribution function of mRNA expression changes in MDA-MB-231 parental cells transiently transfected with mCherry or mCherry∆NLS with genes binned by HNRNPK binding. (D) The empirical cumulative percentages plot of the change in cytoplasmic mRNA. The analysis compares
HNRNPK targets (top 10%, Red, n = 793), (top 20%, Orange, n = 1608), (top 30%, Yellow, n = 2359), (top 40%, Green, n = 3170), and non-targets (Black,n = 1377). (E) The empirical cumulative percentages plot of the change in cytoplasmic 3’UTR mRNA, The analysis compares HNRNPK targets (top 10%,
Red, n = 505), (top 20%, Orange, n = 1026), (top 30%, Yellow, n = 3530), (top 40%, Green, n = 2020), and non-targets (Black, n = 4248). Targets (colored lines)
binned by number of HNRNPK binding sites and non-targets (black line) with minimal gene expression of 4 fragments per kilobase of exon per million
mapped fragments ((FPKM) ≥ 4) are shown

Fallatah et al. BMC Molecular and Cell Biology(2023) 24:26

of K19 loss with cell proliferation. First, we analyzed gene
ontology (GO) pathways for genes that were K19-dependent or downregulated in KRT19 KO cells (Fig. 4A).
Signaling pathways including the Fanconi anemia pathway, regulation of retinoblastoma protein, and direct
p53 effectors were significantly affected in the absenceof K19. Similarly, signaling pathways involving genes
downregulated in HNRNPK KD (Table S6) included
the E2F transcription factor network, Aurora A signaling, Integrin in angiogenesis, and direct p53 effectors(Fig. 4B). Further GO analysis performed using cytoplasmic HNRNPK mRNAs downregulated PAR-CLIP targets
after HNRNPK KD in parental cells (Table S7) showed
direct p53 effectors remained as one of the top enrichedpathways (Fig. 4C). We also performed the same analysis upon overexpressing cytoplasmic HNRNPK in KRT19
KO cells, examining HNRNPK mRNAs via PAR-CLIP for

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all mRNAs (Fig. 4D) and 3’ UTR targets (Fig. 4E, Table
S8) and found that pathways involving p53 were stillamong top 10 affected pathways.
To confirm these observations from GO analyses, we
decided to examine levels of p53 whose mRNA was a
K19- and HNRNPK-dependent cytoplasmic HNRNPK
target (Tables S6, S7). The dysregulation of the p53 pathway by loss of expression or mutation of its components
is one of the major drivers promoting cancer cell survival
and metastasis and the MDA-MB-231 cell line expresses
high levels of the p53 R280K mutant [36]. A link between
HNRNPK and p53 was previously established by reports
documenting that HNRNPK serves as a p53 partner andregulator in response to stress [12, 37]. We decided totest whether K19 loss changes expression levels of p53
or its activator MDM2. We found that p53 and MDM2
levels are decreased in KRT19 KO cells (Fig. 4F). This

Fig. 4 Gene ontology identifies that K19 and HNRNPK promote p53 signaling pathway in MDA-MB-231 cells. (A) The top 10 enriched NCI-Nature pathways for mRNAs downregulated by KRT19 KO. The X axis denotes the number of genes while Y axis denotes NCI-Nature pathways terms. (B) The top 10
enriched NCI-Nature pathways for mRNAs downregulated by HNRNPK KD. The X axis denotes the number of genes while Y axis denotes NCI-Nature
pathways terms. (C) The top 10 enriched NCI-Nature pathways for Cytoplasmic HNRNPK mRNAs downregulated PAR-CLIP targets after HNRNPK KD or(all mRNAs) upon overexpressing cytoplasmic HNRNPK. (D) The top 10 enriched NCI-Nature pathways for Cytoplasmic HNRNPK mRNAs downregulated
PAR-CLIP targets after KRT19 KO or (all mRNAs) upon overexpressing cytoplasmic HNRNPK. (E) Cytoplasmic HNRNPK (3’UTR mRNAs) upon overexpressing
cytoplasmic HNRNPK. The X axis denotes the number of genes while the Y axis denotes NCI-Nature pathways terms. Enriched GO terms among downregulated HNRNPK GO terms for top 10 pathways based on p values are shown. (F) Immunoblots from lysates of Parental MDA-MB-231 and KRT19 KO cellsor (G) Parental and KRT19 KO cells stably expressing GFP or GFP-K19 with antibodies against the indicated proteins. Full-length blots/gels are presented
in Supplementary Figs. 10–11

Fallatah et al. BMC Molecular and Cell Biology(2023) 24:26

decrease was dependent on K19, as the reintroductionof GFP-K19 rescued p53 and MDM2 expression levels(Fig. 4G). Altered p53 pathway gene expression may thus
contribute to the observed decreased cancer cell proliferation accompanying K19 loss.
K19 knockout reduces proliferation of MDA-MB-231 cells

Since mutant p53 was already known to enhance cancer
cell proliferation through binding to other effector proteins like cyclins, we investigated this role after manipulating K19 levels. Previous reports have shown that lossof K19 in estrogen receptor-positive MCF7 breast cancer cells resulted in a delayed cell cycle [21]. We tested
whether K19 also affected the proliferation of MDAMB-231 cells by cell counting and found that KRT19 KO
cells showed significantly decreased cell proliferationcompared to the parental control (Fig. 5A). Similarly,

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KRT19 KO cells showed significantly reduced cell proliferation in a colony formation assay (Fig. 5B, C). We
confirmed the specificity of the KRT19 KO effect on cellproliferation by rescuing K19 expression with a GFPK19 construct (Fig. S6) in our cells, which as expected,
resulted in increased cell proliferation by cell counting(Fig. 5D) and colony formation assays (Fig. 5E, F). Taken
together, our findings show that K19 promotes MDAMB-231 cell proliferation.

Discussion
Overall, we showed that HNRNPK and K19 physicallyinteract in the cytoplasm, and K19 and HNRNPK co-regulate the p53 signaling pathway. We propose that K19 filaments serve as cytoplasmic loading docks for HNRNPK.
Since HNRNPK is a nucleocytoplasmic shuttling protein [18], the interaction between K19 and HNRNPK in

Fig. 5 Cell proliferation of MDA-MB-231 breast cancer cells requires K19. (A) After 50,000 cells were plated on Day 0, cell numbers were counted every
24 h, up to 96 h, using a hemocytometer. (B) 1000 cells were plated on each well of 6 well plates. After growing cells for two weeks, crystal violet staining
was performed to visualize colonies, and images of plates were taken. (C) The colony area was quantified using the ImageJ software. (D) After 50,000
KRT19 KO cells transfected with either GFP or GFP-K19 were plate on Day 0, cell numbers were counted every 24 h, up to 96 h, using a hemocytometer.
(E) 10,000 cells were plated on each well of 6 well plates. After growing cells for two weeks, crystal violet staining was performed to visualize colonies, andimages of plates were taken. (F) The colony area was quantified using the ImageJ software. Data from at least three technical are shown as mean ± SEM.
*** p < 0. 001. Scale bar 0.5 mm

Fallatah et al. BMC Molecular and Cell Biology(2023) 24:26

the cytoplasm and HNRNPK co-sedimentation with K8/
K19 filaments indicate that HNRNPK becomes associated with K8/K19 filaments in the cytoplasm after exiting
the nucleus. Since higher levels of K19 and cytoplasmic
localization of HNRNPK in tumors have been correlatedwith poor patient survival rate and metastasis [8, 38, 39],
our data demonstrate a potential explanation of theseclinical observations on patient survival.
Interestingly, we found that levels of mRNAs withHNRNPK-binding sites in introns were higher inthe presence of K19, supporting the possibility that
HNRNPK generally binds to all its targets first in the
nucleus and then shuttles as part of a larger RNP into
the cytoplasm. In this regard, keratins have been foundinside the nucleus, where they interact with nuclear proteins [40], and K19 contains a bipartite nuclear localization signal towards its C-terminus [40]. Due to its role in
RNA transport and maturation [41, 42], HNRNPK likely
emerges from the nucleus already bound to its targets.
In this scenario, the interaction with K19 would allow
HNRNPK to remain bound to and stabilize its targets
better. Preloading of HNRNPK to its preferred targets
may also explain why the overall number of PAR-CLIPbinding sites was predictive of HNRNPK-mediated regulation of target RNA abundance in the cytoplasm, even
if they were not exclusively found in the cytoplasm, butalso introns.
Using our KRT19 KO system, we identified a comprehensive list of HNRNPK-dependent mRNAs in thecytoplasm. The list includes the E2F transcription factor
network, which we reported to be K19-dependent using
hormone-positive breast cancer cells [21]. Identificationof genes involved in promoting cancer and cell migration is a strong indication that transcriptomic changes of
those genes significantly contribute towards K19-dependent cell proliferation and cytoplasmic HNRNPK-dependent cancer metastasis. Therefore, we have not only
determined how HNRNPK localizes to the cytoplasm
but have also identified novel cytoplasmic RNA targets of
HNRNPK in breast cancer cells.
Given the fact that both K19 and HNRNPK are members of large families of proteins, it is likely that there
are other intermediate filament protein-HNRNP interactions. Indeed, K17 interacts with HNRNPK as mentioned above, and HNRNPS1 proteins have been shownto interact with a type III intermediate filament protein
Table 1 Sequences for Accell Human SMARTpool HNRNPKsiRNAs from Dharmacon
Gene
HNRNPK #1
HNRNPK #2
HNRNPK #3
HNRNPK #4

siRNA sequence
5’-GUAGAGUGCAUAAAGAUCA-3’
5’-CCUAUGAUUAUGGUGGUUU-3’
5’-GCCUUAUGAUCCCAAUUUU-3’
5’-GCAUUUGUAUUGAUAGUUA-3’

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vimentin, which is upregulated in several cancers andplays a critical role in metastasis [43]. Therefore, our findings can be used to characterize the mechanism underlying the progression of various tumor types.

Experimental procedure
Cell culture

The MDA-MB-231 cell line was a generous gift fromDr. Zaver Bhujwalla (Johns Hopkins School of Medicine, Baltimore, MD). MDA-MB-231 cells were authenticated by short-tandem repeat profiling, performed by
ATCC. MDA-MB-231 KRT19 KO cells generated using
the CRISPR/Cas9 system and cells stably expressing
GFP or GFP-KRT19 generated using the lentiviral system were described previously [26]. Stable transductants
were selected using hygromycin (100 µg/ml). Cells weregrown in Dulbecco’s modified essential medium (DMEM;
Gibco, Grand Island, NY) supplemented with 10% Fetal
Bovine Serum (GE Healthcare, Logan, UT) and 1% penicillin/streptomycin (Gibco) in a humidified incubator at
5% CO2 and 37 °C. To measure cell proliferation, 50,000
cells were plated on each well of six-well plates. Cells
were counted using a hemocytometer 24, 48, 72, and 96 h
after plating.
Plasmids and siRNAs

To overexpress HNRNPK, cDNA was cloned out ofpCMV6-AC HNRNPK (OriGene, Rockville, MD) using
the primers described previously [4] and cloned into
pmCherry-C1 (Takara Bio Inc., San Jose, CA). To silence
HNRNPK expression, Accell Human SMARTpool
HNRNPK siRNAs (Dharmacon, Lafayette, CO) was used.
The siRNA sequences targeting HNRNPK are describedin Table 1. AllStars negative control SI03650318 was purchased from Qiagen (Germantown, MD).
Transient transfection

For siRNA, RNAimax lipofectamine (Invitrogen,
Waltham, MA) was used to transfect HNRNPK smartpool siRNA (Dharmacon) or non-targeting siRNA(Qiagen) according to the manufacturer’s protocol. For
overexpression plasmids, jetOptimus DNA transfection
reagent (Polyplus, Illkirch, France) was used according to
the manufacturer’s protocol.
PAR-CLIP analysis

Both parental and KRT19 KO cells were treated with 100µM 4thiouridine (4SU) for 16 h, washed with PBS andirradiated with 0.15 mJ cm2, 365 nm ultraviolet light ina Spectrolinker XL-1500 UV crosslinker to crosslink
RNA to HNRNPK. Cytoplasmic pools of cells were collected by lysing them with a buffer containing 20 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid(HEPES), pH 8, 1 mM ethylenediaminetetraacetic acid

Fallatah et al. BMC Molecular and Cell Biology(2023) 24:26(EDTA), 1.5 mM magnesium chloride, 10 mM potassium
chloride, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl
fluoride, 0.5 mg/mL benzamidine, 0.1 mg/ml leupeptin,
and 1.2 mg/mL aprotinin on ice for 15 min. Then, 7.5 µL
of 10% NP-40 detergent was added to the cells and lysates
were centrifuged for 1 min at 14000 g and 4°C, andsupernatants were collected as cytosolic fractions. 10 U/
ul of RNAse T1 was then added to the lysates followed by
the immunoprecipitation using anti-HNRNPK antibody
and Protein G magnetic beads. The beads were resuspended in one bead volume of dephosphorylation buffer
after washing them with lysis buffer. On beads 3’ adapter
ligation was performed, followed by on beads phosphorylation step. Then, the protein-RNA complexes wereresolved using SDS-PAGE and fluorescent protein-RNA
bands were recovered at the corresponding size ~ 60 kDa.
Proteinase K digestion was performed followed by RNA
recovery by acid phenol/chloroform extraction and ethanol precipitation. After RNA isolation, cDNA library
preparation was carried out [30]. Library preparation
was done using cDNA and sequences by Illumina platform. Obtained reads were processed and referenced tothe hg19 genome assembly (hg19). Data analysis was performed by using PARalyzer settings where T-to-C mutation sequences were filtered. Data from the sequencer
were converted to fastq files by being demultiplexedusing Bcl2fastq (v2.20.0). Adapter barcoded samples were
demultiplexed by using cutadapt (1.15 with python 3.6.4)
with retaining the adapters. Also, libraries were demultiplexed by using 5’ adapter barcode by removing the 5’
adapter barcode anchored to 5’ end of the read. Moreover, data were further processed by PARpipe (https://github.com/ohlerlab/PARpipe) through implementing
Paralyzer as well as annotation of groups and clusters.
Potential PCR duplicates were further removed by allowing the use of additional UMIs in the new protocol andpreserve the true number of reads in the final bam file,during preprocessing fastq sequences were collapsed
prior to adapter and UMI removal [33, 44].
RNA-seq analysis

For RNA-seq analysis, total RNAs from three biological replicates each of untransfected parental and KRT19
KO cells, transfected with siHNRNPK, control siRNA,HNRNPK ∆NLS or vector were extracted. With siHNRNPK, control siRNA was extracted, while withHNRNPK ∆NLS, vector was extracted. Ribosomal RNA
was depleted of using the NEBNext® rRNA Depletion Kit
and cDNA libraries were prepared using the NEBNext®
Ultra™ Directional RNA Library Prep Kit for Illumina®
(NEB, Ipswich MA). RNA was barcoded using the NEBNext Multiplex Oligos for Illumina (NEB). All samples
were multiplexed and sequenced on the Illumina HiSeq

Page 10 of 13

3000 platform using 50 cycles single-end sequencing.
Reads were aligned to hg19 using TopHat2 [45]. Cufflinks
and Cuffdiff were used to quantify transcripts and determine differential expression. The top 500 upregulated
genes with minimum 4 RPKM and p-value 0.05 were
subjected to Enrichr tool, which uses curated gene sets togive biological meaning that can be used for further validation [2].
Antibodies and other reagentsThe following antibodies were used in this study antiGAPDH (FL-335), anti-K19 (A-3), anti-K8 (C51), antiK18 (C-04), anti-PARP (F-2), anti-HNRNPK (3C2) andanti-MDM2 (SMP14) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-p53 (DO-2) was from (MilliporeSigma, St. Louis, MO, USA); anti-GFP (12A6) wasfrom the Developmental Studies Hybridoma Bank (Iowa
City, IA); and anti-RFP (5F8) from ChromoTek (Munich,Germany).
Western blotting

Cells were washed with 1X PBS, and cell lysates were prepared in cold Triton lysis buffer (1% Triton X-100; 40 mm
HEPES, pH 7.5; 120 mm sodium chloride; 1 mm EDTA;
1 mm phenylmethylsulfonyl fluoride; 10 mm sodium
pyrophosphate; 1 µg/ml each of chymostatin, leupeptin,
and pepstatin; 10 µg/ml each of aprotinin and benzamidine; 2 µg/ml antipain; 1 mm sodium orthovanadate; and50 mm sodium fluoride). Cell lysates were centrifuged
to remove cell debris. Protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad) withbovine serum albumin as standard then was prepared in
Laemmli SDS-PAGE sample buffer. Aliquots of protein
lysate were resolved by SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad), cut into smaller pieces
when necessary, and immunoblotted with the indicatedantibodies, followed by horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (MilliporeSigma, St. Louis, MO, USA) and Amersham ECL SelectWestern Blotting Detection Reagent or Pierce ECL Western Blotting Substrate (Thermo Scientific, Hudson, NH).
Signals were detected using ChemiDoc Touch Imager(Bio-Rad) or CL1500 Imaging System (Thermo Fisher
Scientific). For Western blot signal quantitation, theImage Lab software (Bio-Rad) was used.
Biochemical subcellular fractionation

Subcellular fractionation was performed as described
previously [4]. After rinsing with cold 1X PBS, cold
lysis buffer (20 mM HEPES pH 8,1 mM EDTA, 1.5 mM
Magnesium chloride, 10 mM Potassium chloride, 1
mM Dithiothreitol, 1 mM sodium orthovanadate, 1
mM Sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 0.5 mg/mL benzamidine, 0.1 mg/ml leupeptin, andFallatah et al. BMC Molecular and Cell Biology(2023) 24:26

1.2 mg/mL aprotinin) was used to lyse the cells. Then 7.5µL of 10% NP-40 detergent was added to the cells after
incubating them on ice for 15 min. After adding NP-40
detergent to the cells, lysates were centrifuged for 1 min
at 14,000 g and 4 °C, and supernatants were collected as
cytosolic fractions. Pellets were washed 4 times with 1X
PBS and incubated for 40 min at 4 °C (1% Triton X-100,
40 mM HEPES (pH 7.5), 120 mM sodium chloride, 1
mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM
sodium pyrophosphate, 1 µg/ml each of chymostatin,
leupeptin and pepstatin, 10 µg/ml each of aprotinin andbenzamidine, 2 µg/ml antipain, 1 mM sodium orthovanadate, 50 mM sodium fluoride). Supernatants were collected as nuclear fractions after centrifugation for 10 min
at 13,800 g and 4 °C.
Co-immunoprecipitation

Cells were washed with 1X PBS and cell lysates prepared in cold triton lysis buffer (1% Triton X-100; 40 mm
HEPES (pH 7.5); 120 mm sodium chloride; 1 mm EDTA;
1 mm phenylmethylsulfonyl fluoride; 10 mm sodium
pyrophosphate; 1 µg/ml each of chymostatin, leupeptin,
and pepstatin; 10 µg/ml each of aprotinin and benzamidine; 2 µg/ml antipain; 1 mm sodium orthovanadate;
50 mm sodium fluoride) supplemented with 2% empigen
for anti-K19 IP, or cold NP-40 lysis buffer (0.25% NP-40;
50 mM Tris (pH 8.0); 100 mM sodium chloride; 1 mm
phenylmethylsulfonyl fluoride; 10 mm sodium pyrophosphate; 1 µg/ml each of chymostatin, leupeptin, andpepstatin; 10 µg/ml each of aprotinin and benzamidine;
2 µg/ml antipain; 1 mm sodium orthovanadate; 50 mm
sodium fluoride) for anti-HNRNPK IP. Cell lysates were
centrifuged to remove cell debris, and protein concentration was determined using the Bio-Rad Protein Assaywith BSA as standard. Aliquots of cell lysate were thenincubated with the indicated antibody or IgG control,and immune complexes were captured using Protein G
Sepharose (GE Healthcare).
Immunofluorescence (IF) staining

IF staining of cells was performed as described previously [26]. Cells grown on glass coverslips (VWR,
Radnor, PA) were washed with 1X PBS, fixed in 4% paraformaldehyde in 1X PBS for 35 min, and permeabilized
in 0.1% Triton X-100 for 20 min or 0.01% digitonin for
5 min. Samples were blocked in 5% normal goat serum(NGS; RMBIO, Missoula, MT) in 1X PBS before staining with primary antibodies diluted at 1:400 ratio in 5%
NGS blocking buffer and a mixture of 1:1000 of Alexa
Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen) and 1:5000 DAPI (MilliporeSigma, St.
Louis, MO, USA) in 1X PBS was added for 1 h incubation
at RT. After 1X PBS washes, coverslips were mounted on
microscope slides with a mounting medium containing

Page 11 of 13

1,4-diaza-bicyclo[2.2.2]octane (Electron Microscopy
Sciences, Hatfield, PA). Fluorescence images were takenusing the Olympus optical elements fluorescence microscope (Olympus Optical Co., Japan).
Proximity ligation assay

The Duolink in situ proximity ligation assay (PLA) was
performed according to the manufacturer’s protocol(MilliporeSigma, St. Louis, MO, USA). In brief, cells wereplated on glass coverslips, rinsed three times with PBS
and fixed in 3.7% formaldehyde in 1X PBS for 20 min.
The cells were permeabilized in 0.01% digitonin for 5 min
and blocked with 5% NGS in 1X PBS for overnight at
4 °C. After blocking, cells were then incubated with antibodies against HNRNPK, K19 and IgG in 1X PBS containing 5% NGS overnight at 4 °C, followed by incubation
with corresponding secondary antibodies conjugatedwith PLA probes for 60 min at 37 °C in the dark. Cells
were washed three times in 1X PBS. Duolink and DAPI
signals were detected using Olympus optical elements
fluorescence microscope (Olympus). Images are analyzed
using ImageJ [46].
Colony formation assay

Colony formation assay was performed as described previously [25]. On each well of six-well plates, 1,000 cells
were seeded and grown in 2 ml DMEM media for 14
days or 10,000 cells were seeded and grown for 10 days.
The colonies were fixed with 4% formaldehyde and thenstained with 0.5% crystal violet. Images were taken using
ChemiDoc Touch Imager (Bio-Rad, Hercules, CA) andarea of colonies were determined by ImageJ software(National Institutes of Health). Three biological replicateswere analyzed.
Graphs and statistics

All graphs in the manuscript are shown as mean ± standard error of mean. For comparisons between two datasets, a Student’s t test (tails = 2, type = 1) was used, andstatistically significant p-values ≤ 0.05 are indicated in
the figures and figure legends. GraphPad prism and excel
were used to generate graphs in this manuscript.
Abbreviations
HNRNPK	Heterogeneous nuclear ribonucleoprotein
K19	Keratin 19
C-rich	Cytidine-rich
3’UTR	3’ untranslated region
5’UTR	5’ untranslated region
HNRNPK KD	HNRNPK knockdown
KRT19 KO	keratin 19 knockout
KH	K-homology
KI	K interactive
CLIP	crosslinking and immunoprecipitation
K17	Keratin 17
PAR-CLIP	photoactivatable ribonucleoside-enhanced crosslinking andimmunoprecipitation
TNBC	triple negative breast cancer

Fallatah et al. BMC Molecular and Cell Biology(2023) 24:26

RNA-seq	RNA-sequencing
PLA	proximity ligation assays
co-IP	co-immunoprecipitation
K8	Keratin 8
RREs	RNA recognition elements
4SU	4-thiouridine
RNPs	ribonucleoproteins∆NLS	deleted nuclear localization signal
GO	gene ontology

Supplementary Information

Page 12 of 13

Consent for publication
Not applicable.
Disclaimer
This work was prepared while Byung Min Chung was employed at The
Catholic University of America. The opinions expressed in this article are
the author’s own and do not reflect the view of the National Institutes of
Health, the Department of Health and Human Services, or the United States
government.
Received: 28 December 2022 / Accepted: 9 August 2023

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-023-00488-z.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Supplementary Material 5
Supplementary Material 6
Supplementary Material 7
Supplementary Material 8
Supplementary Material 9
Acknowledgments
We thank past and current members of the Coulombe, Chung, and Hafnerlabs for their support and helpful comments. We thank the NIAMS Genomics,
Light Imaging Section, and Flow Cytometry Core Facilities and Gustavo
Gutierrez-Cruz, James Simone, Jiff Lay, Kevin Tinsley, Faiza Naz, and Drs.
Stefania Dell’Orso, Ahsan Polash, and Davide Randazzo (NIAMS/NIH) for
sequencing and flow cytometry support. A preprint of this manuscript can befound in Fallatah, A. (2021). Identifying the Mechanism and Impact of hnRNP K
Regulation by K19. [Doctoral dissertation, The Catholic University of America].
ProQuest LLC.
Authors’ contributions
A.F., M.H. and B.M.C. conceptualized and wrote the main manuscript text. A.F.,
P.S., X.W., A.E, and B.M.C. performed experiments. A.F., D.A., A.M., A.J., S.A., M.H.,
and B.M.C. analyzed results. P.A.C., M.H., and B.M.C., provided resources. M.H.
and B.M.C. supervised and administered the project. All authors reviewed andapproved the manuscript.
FundingNational Cancer Institute grant R15CA213071 (B.M.C.); National Institutefor Arthritis, Musculoskeletal and Skin diseases grant R01AR044232 (P.A.C.);
and Intramural Research Programs of the National Institute for Arthritis andMusculoskeletal and Skin Diseases (NIAMS to M.H) took part in the design ofthe study and collection, analysis, interpretation of data, and the writing of themanuscript.
Data availability
All data generated or analyzed during this study are included in thispublished article, its supplementary information files, and the GEO repository,
GSE223603.

Declarations
Competing interests
The authors declare no competing interests.
Ethics approval and consent to participate
Not applicable.

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