Skip to main content
NCBI home page
The FASEB Journal logoLink to The FASEB Journal
. 2011 Jul;25(7):2305–2312. doi: 10.1096/fj.10-177808

A transcribed pseudogene of MYLK promotes cell proliferation

Yoo Jeong Han *,†, Shwu Fan Ma *, Gregory Yourek †, Yoon-Dong Park ‡, Joe G N Garcia ‡,§,1
PMCID: PMC6137860  PMID: 21441351

Abstract

Pseudogenes are considered nonfunctional genomic artifacts of catastrophic pathways. Recent evidence, however, indicates novel roles for pseudogenes as regulators of gene expression. We tested the functionality of myosin light chain kinase pseudogene (MYLKP1) in human cells and tissues by RT-PCR, promoter activity, and cell proliferation assays. MYLKP1 is partially duplicated from the original MYLK gene that encodes nonmuscle and smooth muscle myosin light chain kinase (smMLCK) isoforms and regulates cell contractility and cytokinesis. Despite strong homology with the smMLCK promoter (∼89.9%), the MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. Moreover, MYLKP1 and smMLCK exhibit negatively correlated transcriptional patterns in normal and cancer cells with MYLKP1 strongly expressed in cancer cells and smMLCK highly expressed in non-neoplastic cells. For instance, expression of smMLCK decreased (19.5±4.7 fold) in colon carcinoma tissues compared to normal colon tissues. Mechanistically, MYLKP1 overexpression inhibits smMLCK expression in cancer cells by decreasing RNA stability, leading to increased cell proliferation. These studies provide strong evidence for the functional involvement of pseudogenes in carcinogenesis and suggest MYLKP1 as a potential novel diagnostic or therapeutic target in human cancers.—Han, Y. J., Ma, S. F., Yourek, G., Park, Y.-D., Garcia, J. G. N. A transcribed pseudogene of MYLK promotes cell proliferation.

Keywords: myosin light chain kinase, human cancers, gene duplication, RNA stability

Pseudogenes, originally derived from paralogous functional genes, are generally defunct because of either the lack of regulatory elements or the presence of frameshift mutations (1–2). As a result of the high level of sequence similarity to the corresponding functional genes, pseudogenes often pose a challenge for gene prediction programs, with frequent misidentification as real genes. For instance, initial interpretation of the sequence data from human chromosome 22 indicated that 19% of the coding sequences were pseudogenic (3). Several direct surveys of pseudogenes using more robust methodologies, however, revealed that the estimated number of pseudogenes is ∼20,000 (4–5), a figure comparable to the number of protein-coding genes (∼27,000) in the human genome (6). Despite the abundance of pseudogenes in the human genome, the pathophysiological roles of pseudogenes remain poorly understood.

Duplicated pseudogenes represent one major type of pseudogene and arise from tandem duplication of genomic DNAs or unequal crossing over. In contrast, retrotransposed pseudogenes, the other major type of pseudogene, result from the reverse transcription of mRNA transcript followed by integration into the genomic DNA, a process known as retrotransposition (5). Duplicated pseudogenes usually retain the original or partial exon-intron structures of the functional genes but with multiple degenerative mutations that drive premature stop codons (7). Certain pseudogenes, however, are potentially transcriptionally active, expressing mRNAs utilizing their own promoters or adjacent promoters (8–9). In fact, duplicated pseudogenes have been reported to produce antisense RNA and inhibit functional gene expression through an antisense-sense mechanism (10). Retrotransposed pseudogenes, in contrast, are often considered as a special type of retrotransposon, which consists of repetitive sequences, such as Alu or LINEs (long interspersed nuclear elements). In the process of retrotransposition, these pseudogenes usually contain a poly-A tail and generally lack introns, a hallmark of cDNAs (2). Recent data demonstrated that retrotransposed pseudogenes generate naturally occurring small interfering RNA (siRNA) and Piwi-interacting RNA (piRNA) in mouse oocytes (11).

As these findings suggest active biological roles of pseudogenes in regulating gene expression, we were interested in the myosin light chain kinase pseudogene (MYLKP1; HGNC ID:7591), an intrachromosomal duplication of exons 13–17 of myosin light chain kinase gene (MYLK, HGNC ID:7590) (12). MYLK is an intricate gene spanning >270 kb and containing 34 exons on 3q13–q21 of human chromosome 3 (13). By alternative splicing (14), MYLK generates at least 9 transcripts that encode 3 proteins, including a 220-kDa nonmuscle myosin light chain kinase (nmMLCK), a 130-kDa smooth muscle MLCK (smMLCK; ref. 15), and a 20-kDa protein known as telokin. The MYLK pseudogene and functional MYLK share high levels of similarity (93%) in their DNA sequences. MLCK is a fundamental regulator of actin-myosin interaction via phosphorylation (catalyzed by MLCK in the presence of Ca2+/calmodulin) and dephosphorylation (catalyzed by myosin phosphatase 1) of the 20-kDa regulatory light chain of myosin-II (MLC20) (16). Both smMLCK and nmMLCK are essential participants in many biological processes, including cell motility (17), cytokinesis (18), the timing of mitosis (19), and apoptosis (20), and they are critical to key pathophysiological features of human diseases, including essential hypertension (15, 21–22), as well as cardinal features of the inflammatory response, including vascular permeability and inflammatory cell trafficking (23).

Expression of genes that encode cytoskeletal proteins, such as MLCK, are regulated in angiogenesis and in tumors that exhibit increased invasiveness (24). We hypothesized that MYLKP1 may play a significant biological role in neoplastic processes and now show that MYLKP1 selectively transcribes mRNA in cancer cells and dramatically decreases the expression of the functional MYLK. Moreover, expression of the pseudogene increases cell proliferation of normal cells, indicating an active role of MYLKP1 during carcinogenesis. These studies, which provide further support for the functional involvement of pseudogenes in human pathobiology, suggest that MYLKP1 should be considered a novel diagnostic or therapeutic target in human cancer.

MATERIALS AND METHODS

In silico analyses and molecular cloning

The transcription start site (TSS) for smMLCK was identified by the publicly available Cap-Analysis Gene Expression (CAGE) database (http://fantom3.gsc.riken.jp) and validated through the Genomatix database (http://www.genomatix.de). On the basis of the analyses, proximal promoter regions of smMLCK (−876 to +1) and MYLKP1 (−834 to +1) (see Fig. 2) were amplified by PCR using a MYLK Bac clone (RP11-gn20) and a MYLK pseudogene cos82 cosmid as templates. Detailed primers sequences are shown in Supplemental Table S1. Respective amplicons were then cloned into a pGL3-Basic Firefly luciferase vector at KpnI and HindIII sites. For the cloning of coding sequences of MYLKP1, the targeting region was amplified from the cos82 (illustrated in Fig. 5A) using the primer pairs (Supplemental Table S1). These products were inserted into BamH I and XbaI sites of the pcDNA 3.1. The integrity of these constructs was confirmed by DNA sequencing using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) in the Cancer Research Center at University of Chicago.

Figure 2.

Figure 2.

Open in a new tab

DNA sequences and activity of MYLKP1 promoter. A) Exonic and intronic structures of MYLK. Transcription of smMLCK begins in intron 16 (indicated by arrow); translation (ATG) starts in exon 17. B) Proximal region of MYLKP1 promoter was sequenced and compared with sequences of the smMLCK promoter. Mismatched sequences are indicated in red; exon sequences are capitalized. C) BEAS-2b cells transfected with MYLKP1 promoter exhibited lower luciferase activity compared to cells transfected with the smMLCK promoter. D) LAC-H522 cells transfected with the MYLKP1 promoter showed similar luciferase activity as cells transfected with the smMLCK promoter. Each experiment was repeated 4 times; results are reported as means ± sd.

Figure 5.

Figure 5.

Open in a new tab

Expression of the MYLK pseudogene inhibits protein expression of the functional MYLK and stimulates cell proliferation. A) HUFs were transfected with vehicle only (lane 1), pcDNA 3.1 (lane 2), and exon 17 of MYLKP1 in the reading frame (lane 3) or out of the reading frame (lane 4). RT-PCR analysis showed that expression of MYLKP1 inhibits endogenous MLCK expression regardless of its frame. B) Western blot analysis showed that expression of MYLKP1 inhibits protein expression of smMLCK (lanes 2, 3). C) Cells expressing MYLKP1 show an increase in cell proliferation compared to cells expressing pcDNA 3.1. Cell numbers were counted in MD-AMB-231 cells transfected with pcDNA 3.1 or MYLKP1 clone 3 up to 5 d. Experiment was repeated 3 times; results are reported as means ± sd. *P < 0.05 vs. pcDNA3.1.

Cell cultures

Primary cultured human uterine fibroblasts (HUFs) were provided by Dr. Zuzana Strakova (University of Illinois at Chicago) and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated and charcoal-stripped FBS, and 0.1 mM sodium pyruvate. Breast cancer cell lines were provided by Dr. Olufunmilayo Olopade (University of Chicago), and additional cell lines were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured according to the manufacturer's protocol.

Reporter activity assays and RNA stability assays

For dual luciferase reporter gene assays, cells grown in 12-well plates were cotransfected with 1 μg of the firefly luciferase vector containing smMLCK or MYLKP1 promoter and 20 ng of TK-renilla luciferase vector (Promega, Madison, WI, USA) using Fugene HD transfection reagent (Roche, Basel, Switzerland) as described previously (15). For coexpression of smMLCK and MYLKP1, cells grown in 60-mm dishes were cotransfected with the fixed amount (3 μg) of smMLCK expression vector and specific various amounts (0 to 3 μg) of MYLKP1 clone 3 vector utilized to test a dose effect of MYLKP1 expression on smMLCK. For RNA stability assays, cells were cotransfected with 3 μg of smMLCK expression vector and 0.5 μg of pcDNA 3.1 or 0.5 μg of MYLKP1 expression vector using Fugene 6 transfection reagent. At 16 h after transfection, cells were treated with 10 μg/ml actinomycin D for 5 or 9 h and harvested for RNA isolation.

RNA isolation, RT-PCR, and miRNA array profiling

Total RNA was purchased from Agilent Technologies (Santa Clara, CA, USA) or isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. For a conventional RT-PCR, each reaction was carried out with 2 μl cDNA, 0.5 μM forward (3bf) and reverse (3ar) primers (12), and 0.01 U Phusion DNA polymerase (Finnzymes, Espoo, Finland). Three-step PCR was performed according to the manufacturer's protocol, but only 22–25 cycles were executed for semiquantitative analysis with a primer labeled with 32P-ATP or 28–30 cycles with unlabeled primers. The signal was detected by autoradiography after 6% PAGE or by ethidium bromide staining after 2% agarose gel. The lower band shown on agarose gels (see Fig. 4A, lane 1) corresponding to MYLKP1 fragment was isolated, sequenced, and verified. Quantitative RT-PCR was performed according to manufacturer's protocol using SYBR Green PCR Master Mix (Applied Biosystems), and the primers were targeted at exon 23 (Supplemental Table S1), as illustrated in Fig. 3A. The specificity of primers was validated by a dissociation curve analysis, and the fold change in expression of each gene was calculated using the ΔΔCt method, with 18S rRNA as an internal control. For miRNA array profiling, Exiqon miRCURY LNA arrays (Exiqon, Vedbaek, Denmark) were used, following the manufacturer's protocol.

Figure 4.

Figure 4.

Open in a new tab

Expression of the MYLK pseudogene inhibits mRNA expression of smMLCK. A) MYLK pseudogene is transcribed in human cancers. RT-PCR was performed using total RNA isolated from normal human tissues (breast, brain, colon, cervix, lung, liver, uterus, and vein), normal human cells (human aortic smooth muscle cells, human embryonic kidney 293 cells, and human mammary epithelial MCF-10A cells), human carcinoma tissues (bladder carcinoma, colon carcinoma, lymphoma, and vulvar carcinoma) and human cancer cell lines (human promyelocytic leukemia HL-60 cells, human endometrial adenocarcinoma ECC-1 cells, human cervical cancer HeLa cells, human breast cancer MCF-7 cells, and human colon cancer SW620 cells). Genomic DNA (lane 1) was used as control and showed 2 distinct amplicons of MYLK and MYLKP1 with a 72-bp difference. MYLK pseudogene specifically transcribes in cancer cells/tissues. B) Clones were constructed with various regions of MYLKP1. Clone 1: exon 16 to exon 17; clone 2: intron 16 to exon 17; clone 3: exon 17; clone 4: ATG to the end of exon 17; and clone 5: exon 16 and intron 16. C) MYLK and MYLKP1 expression were detected using RT-PCR in BEAS-2b transfected with the various constructs shown in B. Mock (pcDNA 3.1) is used as positive control. D) RT-PCR detecting expression level of MYLK and MYLKP1 in cells cotransfected with smMLCK and MYLKP1 constructs. Numbers indicate amount (μg) of plasmid used for cotransfection. Each experiment was repeated 3 times; representative data are shown.

Figure 3.

Figure 3.

Open in a new tab

Level of functional MYLK transcripts decreased in human cancers. A) Schematic diagram of structure of nmMLCK, smMLCK, telokin, and MYLKP1 transcripts. Rectangular box indicates location of primers (exon 23) designed for qRT-PCR, which detected both nmMLCK and smMLCK transcripts but not MYLKP1 or telokin transcript. B) qRT-PCR was performed with RNA isolated from various tissues with or without carcinomas. Total MLCK (nmMLCK and smMLCK) expression is notably lower in carcinoma tissues compared to normal tissues. C, D) mRNA expression was compared in the same type of tissues: lymph node vs. lymphoma (C) or colon vs. colon carcinoma (D). E) RNA was isolated from normal cells or cancer cell lines. Expression of nmMLCK and smMLCK isoforms is remarkably lower in cancer cells compared to normal cells. Each experiment was repeated 6 times; results are reported as means ± sd.

Western blot analysis and cell proliferation assays

Western blot was performed using an anti-smMLCK antibody, as described previously (22). For proliferation assays, cells were transfected with pcDNA 3.1 or MYLKP1 clone 3 using Fugene HD transfection reagent (Roche). Two days after transfection, cells were selected with 400 μg/ml of Geneticin (G418; Sigma-Aldrich, St. Louis, MO, USA) and maintained with 200 μg/ml of G418. Cells grown in a 12-well plate with initial number of 105 cells/well were harvested each day and counted using Countess Automated Cell Counter (Invitrogen) up to 5 d.

RESULTS

Genomic structure analyses of MYLK and the MYLK pseudogene

The locations of the functional MYLK and MYLKP1 on chromosome 3q13–3q21 and 3p12 were verified in prior reports (12–13) and stored in the National Center for Biotechnology Information and Ensembl databases. As noted above, MYLK gene contains 34 exons that encode 3 proteins (nmMLCK, exons 4–34; smMLCK, exons 17–34; and telokin, exons 32–34) with each protein regulated independently by individual promoters with unique TSSs (15, 25–26). In contrast, MYLKP1 contains 5 exons, which overlap with exons 13–17 of the functional MYLK gene (Fig. 1). DNA sequence analysis reveals that MYLKP1 contains a promoter region in intron 16 that is 89.9% identical to the functional smMLCK promoter. Detailed alignment and DNA sequences of the promoter region are shown in Fig. 2A, B.

Figure 1.

Figure 1.

Open in a new tab

Location and structure of MYLK and MYLKP1 in the human genome. Functional MYLK is located on chromosome 3q21 and produces 3 isoforms: nonmuscle MLCK (exons 4–34), smooth muscle MLCK (exons 17–34), and telokin (exons 32–34). The gene is partially duplicated and relocated on chromosome 3p12, resulting in a MYLK pseudogene, MYLKP1, containing only exons 13–17. MYLKP1 has a promoter region similar to the smooth muscle MLCK promoter in intron 16.

MYLK pseudogene promoter activities

To determine whether the MYLKP1 promoter shares functional similarity with the smMLCK promoter, the proximal region of smMLCK and MYLKP1 promoters were amplified and cloned into pGL3-Basic (pGL3B) firefly luciferase reporter vectors. The promoter-luciferase constructs were transfected into normal bronchial epithelial (BEAS-2b) cells or lung adenocarcinoma (LAC-H522) cells. Reporter gene activity assays showed that MYLKP1 promoter activity was extremely reduced in normal cells compared to the activity of the smMLCK promoter (Fig. 2C). However, MYLKP1 promoter activity was increased in lung adenocarcinoma cells, achieving a level comparable to smMLCK promoter activity (Fig. 2D), indicating that the activity of the MYLKP1 promoter is potentially functionally specific to cancer cells.

MLCK expression is reduced in human cancers

Quantitative RT-PCR (qRT-PCR) was performed to determine the expression level of functional MYLK using total RNA isolated from 8 different types of normal tissues, 4 types of carcinoma tissues, 3 types of normal cells, and 4 types of cancer cell lines. As DNA sequences from exons 17–34 are identical in both nmMLCK and smMLCK, primers designed specifically to target exon 23 detect the transcripts of nmMLCK and smMLCK but do not detect MYLKP1 or telokin (Fig. 3A). MLCK expression is tissue type dependent (Fig. 3B); however, MLCK expression was significantly reduced in neoplastic carcinoma tissues compared to normal tissues (Fig. 3B). These differences in MLCK expression were more obvious when comparisons of normal vs. neoplastic tissues included tissues from identical anatomic sites, with significantly reduced MLCK expression in lymphomatous tissues compared to lymph nodes (5.14±0.98-fold decrease; Fig. 3C) and dramatically reduced in colon carcinomas compared to normal colon tissues (19.53±4.7 fold decrease; Fig. 3D). Despite varying levels of MLCK expression in different cell types, MLCK expression was consistently and significantly reduced in cancer cell lines compared to normal nonneoplastic cells (Fig. 3E).

MYLK pseudogene transcribes in human cancers

To determine the transcriptional activity of MYLKP1 in normal and cancer cells, we first analyzed DNA sequences of exon 17 of smMLCK and MYLKP1 (Supplemental Fig. S1A). Major differences were identified, including a single-nucleotide deletion at position 121 and a 72-bp deletion (nt 342–413) observed in MYLKP1. The single-nucleotide deletion drives a frameshift resulting in multiple-stop codons in MYLKP1 (Supplemental Fig. S1B). The transcriptional activity of MYLKP1 was examined by performing a RT-PCR using the primers 3ar and 3bf (12), designed from the common regions of smMLCK and MYLKP1 (Supplemental Fig. S1A). However, the amplicon size for MYLKP1 is smaller (594 bp) than for MYLK (667 bp) due to the 72-bp deletion in MYLKP1 (Supplemental Fig. S1A). RT-PCR data clearly showed that only MYLK is transcribed in normal tissues and cells (regardless of cell and tissue origins; Fig. 4A, lanes 2–12), whereas MYLKP1 is actively and differentially transcribed in diverse neoplastic tissues and cancer cell lines (Fig. 4A, lanes 13–21).

MYLK pseudogene inhibits RNA expression of functional MLCK in cancers

As our studies clearly demonstrated that functional MYLK expression is decreased in cancer cells and tissues (Fig. 3), whereas pseudogene expression is increased (Fig. 4A), we next tested whether the transcriptional activity of functional MYLK was inhibited by MYLKP1 in cancers. Five MYLKP1 fragments corresponding to various regions of MYLKP1 (named clones 1–5) were amplified and subcloned into pcDNA3.1 vector (Fig. 4B) to determine which MYLKP1 region is responsible for inhibition of functional MYLK expression. MYLKP1 clones and vector alone (pcDNA) were transfected into BEAS-2b cells, and RT-PCR was performed using the same primer pair (3bf and 3ar). Cells transfected with pcDNA produced a 667-bp amplicon, again indicating that MYLK is the only gene expressed in BEAS-2b (Fig. 4C). Transfection of BEAS-2b cells with MYLKP1 clones 1–4 resulted in complete inhibition of endogenous MYLK expression, whereas MYLKP1 expression (corresponding to the 594-bp amplicon) was significantly increased (Fig. 4C, lanes 2–5). Clone 5, containing exon 16 and intron 16, failed to inhibit the transcriptional activity of MYLK (Fig. 4C, lane 6), indicating that exon 17, the first exon downstream of the promoter region, is crucial for inhibition of functional MYLK expression. To further examine potential inhibitory effects of MYLKP1, we coexpressed the fixed amount (3 μg) of smMLCK with increasing amounts (0–3 μg) of MYLKP1 clone 3 (Fig. 4D). BEAS-2b cells transfected with smMLCK alone were used as a control (Fig. 4D, lane 1). Stepwise increases in MYLKP1 expression resulted in reduced smMLCK expression (Fig. 4D, lanes 2–4) with >90% inhibition at the 1:1 ratio of MYLKP1 to smMLCK (Fig. 4D, lane 4). These results clearly demonstrated a potent inhibitory effect of MYLKP1 on smMLCK expression.

MYLK pseudogene functions regardless of reading frames

To determine whether amino acid codons of MYLKP1 are important for the inhibitory effect, we transfected primary cultured HUFs with 2 additional MYLKP1 constructs designed to express exon 17 either in or out of the reading frame (Fig. 5). RT-PCR was performed using 32P-labeled primers, and the products were subjected to 6% PAGE and autoradiography. These experiments showed that MYLKP1 clones, including out-of-frame constructs, completely inhibit RNA expression of endogenous smMLCK regardless of their frames (Fig. 5A). These results are consistent with the notion that amino acid codons of MYLKP1 are not essential for inhibition of functional smMLCK expression. Similarly, the presence of premature stop codons (Supplemental Fig. S1B) is also irrelevant for MYLKP1 inhibitory function, as all MYLKP1 clones containing the stop codons exhibit inhibitory effects on MLCK expression (Figs. 4C and 5A).

MYLK pseudogene inhibits MLCK protein expression and stimulates cell proliferation

We utilized Western blots to determine functional MLCK protein expression in HUFs transfected with MYLKP1 (Fig. 5B). Expression of MYLKP1, whether in or out of the reading frame, significantly decreased smMLCK protein expression (Fig. 5B, lanes 2, 3) compared to cells transfected with pcDNA (lane 1). MYLKP1 expression decreased both mRNA (Fig. 5A) and protein expression (Fig. 5B) of the functional MYLK. We next investigated the potential for MYLKP1 to drive cell proliferation, a strong neoplastic characteristic, and monitored proliferation rates of MD-AMB-231 cells stably transfected with pcDNA3.1 or MYLKP1 clone 3. MYLKP1 significantly increased MD-AMB-231 cell proliferation at d 4 (3.5± 0.46×105 cells vs. 2.2±0.02×105 cells transfected with pcDNA 3.1; Fig. 5C).

MYLK pseudogene transcribes a sense strand and decreases RNA stability of MYLK

Because MYLKP1 expression inhibits the functional MYLK gene expression at the mRNA level, we investigated potential mechanisms of the pseudogene effects on mRNA expression of MYLK, including the possibility that the MYLKP1 transcript acts as an antisense mRNA interfering with a sense mRNA transcribed from the functional MYLK. To test the possibility, we synthesized first-strand cDNA using either a sense-strand or an antisense-strand specific primer, digested RNA templates, and amplified cDNA by PCR using 2 primers shown in Supplemental Fig. S1A. The data clearly show that MYLKP1 as well as MYLK transcribes the sense strand (Fig. 6A, lanes 3, 5), not the antisense strand (Fig 6A, lanes 4, 6). We next investigated whether MYLKP1 encodes a miRNA that potentially interferes with functional MYLK expression. We profiled miRNAs using Exiqon miRCURY LNA arrays with total RNA isolated from BEAS-2b or lung adenocarcinoma cells transfected with pcDNA, MYLKP1, or smMLCK. Significant changes in miRNA profiles were not detected in cells transfected with MYLKP1 when compared to cells transfected with pcDNA or smMLCK. Lastly, we determined whether MYLKP1 decreases MYLK expression at the post-transcriptional level by regulating RNA stability. Cells were cotransfected with smMLCK and pcDNA (Fig. 6B, lanes 1–3) or smMLCK and MYLKP1 (Fig. 6B, lanes 4–6), treated with actinomycin D for 5 or 9 h, and subjected to RNA isolation and RT-PCR. For this assay, we used a 6:1 ratio of smMLCK to MYLKP1 to attain a partial inhibition of smMLCK expression by MYLKP1. In cells cotransfected with smMLCK and pcDNA (Fig. 6B, lanes 1–3), 9 h treatment of actinomycin D resulted in only a 22% reduction in the amount of MYLK mRNA (Fig. 6B, lane 3) compared to no treatment control (Fig. 6B, lane 1). In contrast, in cells cotransfected with smMLCK and MYLKP1, the same treatment drove 91% reduction in the amount of MYLK mRNA (Fig. 6B, lane 6) compared to no treatment control (Fig. 6B, lane 4). These data show that coexpressing MYLKP1 with smMLCK decreased mRNA stability of smMLCK. Taken together, MYLKP1 is specifically transcribed in cancer cells, regulates functional MYLK expression at the post-transcriptional level, and stimulates cell proliferation in human cancers.

Figure 6.

Figure 6.

Open in a new tab

MYLK pseudogene transcribes a sense strand and decreases RNA stability of MYLK. A) RNA was isolated from normal cells (BEAS-2b) or lung adenocarcinoma cells and synthesized to first cDNA using a strand-specific primer followed by PCR amplification. Normal cells transcribe the sense strand of MYLK (lane 3); lung adenocarcinoma cells transcribe the sense strand of MYLKP1 (lane 5). B) Cells cotransfected with smMLCK and pcDNA (lanes 1–3) or smMLCK and MYLKP1 (lanes 4–6) were treated with actinomycin D (ActD) for 5 or 9 h. Coexpression of MYLKP1 significantly decreases the level of MYLK mRNA (lane 6). Experiment was repeated 3 times; results are reported as means ± sd.

DISCUSSION

Since their discovery more than 3 decades ago (7, 27), pseudogenes have generally been considered to represent nonfunctional artifacts of evolutionary processes due to degenerative features, such as the absence of promoters or the presence of premature stop codons. Increasing recent evidence, however, now indicates potentially novel pathophysiological roles for pseudogenes as regulators of gene expression via generation of siRNAs (11, 28), antisense RNAs (10, 29), or regulation of RNA stability (30). In this study, we provide novel evidence demonstrating the functionality of the pseudogene MYLKP1, partially duplicated from MYLK during the evolution to high primates, with a pathophysiological role in human cancers. The MYLKP1 promoter is selectively activated in neoplastic processes (Fig. 2C, D), with MYLKP1 mRNA expressed in human cancer cells and tissues, repressed in normal cells and tissues (Fig. 4A), and regulated in cell proliferation during carcinogenesis (Fig. 5C).

Pseudogenes are effectively genetic fossils capturing genes in the past and providing precious clues about genome dynamics, such as gene duplication events (for duplicated pseudogenes) and retrotransposition events (for processed pseudogenes). Gene duplication events likely occur at a constant and high rate in humans, as indicated by the abundance of copy-number variants. Duplicated pseudogenes are “unsuccessful duplicates” inactivated by degenerative mutations, whereas “successful duplicates” lead to copy-number variations among individuals (31). Interestingly, the majority of the unsuccessful duplication affected specific gene functional categories, the protein families associated with environmental responses (32). For instance, certain cell-surface protein pseudogenes are reactivated when yeast is challenged by a stressful new environment, indicating that pseudogenes are conditionally activated in response to specific signals (32). These conceptual underpinnings are consistent with our finding that the MYLKP1 promoter is selectively activated in neoplastic processes reflected by silent MYLKP1 promoter activities in normal cells but markedly active promoter activities in cancer cells (Fig. 2C, D). The difference appears to be imprinted within DNA sequences; despite 89.9% identity between MLCKP1 and smMLCK promoters, the MLCKP1 promoter contains 3 deletion mutations and 54 point mutations within a 876-bp region of smMLCK promoter (Fig. 2B). The absence of cis-acting elements produced by these deletions would conceivably inactivate the promoter under normal conditions. However, cancer-specific transcription factors could potentially overcome the absence of these elements and reactivate the MYLKP1 promoter under specific conditions. Indeed, in silico analysis showed that the deletion mutations caused the absence of several important transcription factor binding sites, whereas point mutations created new elements for cancer-specific transcription factors. While the exact mechanistic details for MYLKP1 promoter regulation remain to be elucidated, our data demonstrated the selective activation of the MYLKP1 promoter in neoplastic processes. Along with MYLKP1, other pseudogenes might play important roles during neoplastic processes, because the human genome contains several tumor suppressor pseudogenes, such as p53 pseudogene (33), PTEN pseudogene (34), and breast cancer 1 (BRCA1) pseudogene (35). Although their functional roles remain unclear, the identification of these pseudogenes has important implications for the understanding of biological processes of carcinogenesis.

In summary, we describe a novel role of a MYLKP1 pseudogene specifically expressed in cancerous conditions and that stimulates cell proliferation. Because MYLKP1 is present only in high primates, such as humans, chimpanzees, and gorillas, this unique pseudogene serves as a potentially unique characteristic of human cancers, distinguished from the common traits of cancers in model animals. Considering the abundance of pseudogenes in the human genome, studying the roles of pseudogenes in human cancers may elucidate more precise mechanisms of human neoplasia, develop novel biomarker to diagnose human cancers, and eventually lead to a greater understanding of the role of pseudogenes in human health and disease.

Supplementary Material

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

Click here for additional data file. (26KB, doc)
Click here for additional data file. (8.3MB, tif)
Click here for additional data file. (11.4KB, docx)

Acknowledgments

The authors thank Dr. Joseph A. Piccirilli (University of Chicago) for critically reviewing the manuscript, Dr. Dominique Giorgi (Institut de Genetique Humaine, Montpellier, France) for generously providing the cos82 cosmid vector containing the MYLK pseudogene, Dr. Zuzana Strakova (University of Illinois at Chicago) for HUFs, and Dr. Olufunmilayo Olopade (University of Chicago) for breast cancer cell lines. The authors also thank their colleagues Dr. Jaideep Moitra (University of Chicago) for providing the MYLK Bac clone, Dr. Djanybek Adyshev (University of Chicago) for the MYLK cDNA clone, and Dr. Frances Lennon and Dr. Patrick Singleton (University of Chicago) for lung adenocarcinoma cells.

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

REFERENCES

  • 1.Balakirev E. S., Ayala F. J. (2003) Pseudogenes: are they “junk” or functional DNA?, Annu. Rev. Genet. , 123–151 [DOI] [PubMed] [Google Scholar]
  • 2.Zhang Z., Harrison P. M., Liu Y., Gerstein M. (2003) Millions of years of evolution preserved: a comprehensive catalog of the processed pseudogenes in the human genome, Genome Res. , 2541–2558 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Dunham I., Shimizu N., Roe B. A., Chissoe S., Hunt A. R., Collins J. E., Bruskiewich R., Beare D. M., Clamp M., Smink L. J., Ainscough R., Almeida J. P., Babbage A., Bagguley C., Bailey J., Barlow K., Bates K. N., Beasley O., Bird C. P., Blakey S., Bridgeman A. M., Buck D., Burgess J., Burrill W. D., Burton J., Carder C., Carter N. P., Chen Y., Clark G., Clegg S. M., Cobley V., Cole C. G., Collier R. E., Connor R. E., Conroy D., Corby N., Coville G. J., Cox A. V., Davis J., Dawson E., Dhami P. D., Dockree C., Dodsworth S. J., Durbin R. M., Ellington A., Evans K. L., Fey J. M., Fleming K., French L., Garner A. A., Gilbert J. G. R., Goward M. E., Grafham D., Griffiths M. N., Hall C., Hall R., Hall-Tamlyn G., Heathcott R. W., Ho S., Holmes S., Hunt S. E., Jones M. C., Kershaw J., Kimberley A., King A., Laird G. K., Langford C. F., Leversha M. A., Lloyd C., Lloyd D. M., Martyn I. D., Mashreghi-Mohammadi M., Matthews L., McCann O. T., McClay J., McLaren S., McMurray A. A., Milne S. A., Mortimore B. J., Odell C. N., Pavitt R., Pearce A. V., Pearson D., Phillimore B. J., Phillips S. H., Plumb R. W., Ramsay H., Ramsey Y., Rogers L., Ross M. T., Scott C. E., Sehra S. K., Skuce C. D., Smalley S., Smith M. L., Soderlund C., Spragon L., Steward C. A., Sulston J. E., Swann R. M., Vaudin M., Wall M., Wallis J. M., Whiteley M. N., Willey D., Williams L., Williams S., Williamson H., Wilmer T. E., Wilming L., Wright C. L., Hubbard T., Bentley D. R., Beck S., Rogers J., Shimizu N., Minoshima S., Kawasaki K., Sasaki T., Asakawa S., Kudoh J., Shintani A., Shibuya K., Yoshizaki Y., Aoki N., Mitsuyama S., Roe B. A., Chen F., Chu L., Crabtree J., Deschamps S., Do A., Do T., Dorman A., Fang F., Fu Y., Hu P., Hua A., Kenton S., Lai H., Lao H. A., Lewis J., Lewis S., Lin S.-P., Loh P., Malaj E., Nguyen T., Pan H., Phan S., Qi S., Qian Y., Ray L., Ren Q., Shaull S., Sloan D., Song L., Wang Q., Wang Y., Wang Z., White J., Willingham D., Wu H., Yao Z., Zhan M., Zhang G., Chissoe S., Murray J., Miller N., Minx P., Fulton R., Johnson D., Bemis G., Bentley D., Bradshaw H., Bourne S., Cordes M., Du Z., Fulton L., Goela D., Graves T., Hawkins J., Hinds K., Kemp K., Latreille P., Layman D., Ozersky P., Rohlfing T., Scheet P., Walker C., Wamsley A., Wohldmann P., Pepin K., Nelson J., Korf I., Bedell J. A., Hillier L., Mardis E., Waterston R., Wilson R., Emanuel B. S., Shaikh T., Kurahashi H., Saitta S., Budarf M. L., McDermid H. E., Johnson A., Wong A. C. C., Morrow B. E., Edelmann L., Kim U. J., Shizuya H., Simon M. I., Dumanski J. P., Peyrard M., Kedra D., Seroussi E., Fransson I., Tapia I., Bruder C. E., O'Brien K. P. (1999) The DNA sequence of human chromosome 22. Nature , 489–495 [DOI] [PubMed] [Google Scholar]
  • 4.Harrison P. M., Hegyi H., Balasubramanian S., Luscombe N. M., Bertone P., Echols N., Johnson T., Gerstein M. (2002) Molecular fossils in the human genome: identification and analysis of the pseudogenes in chromosomes 21 and 22, Genome Res. , 272–280 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Goncalves I., Duret L., Mouchiroud D. (2000) Nature and structure of human genes that generate retropseudogenes, Genome Res. , 672–678 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature , 931–945 [DOI] [PubMed] [Google Scholar]
  • 7.Proudfoot N. (1980) Pseudogenes. Nature , 840–841 [DOI] [PubMed] [Google Scholar]
  • 8.Endoh A., Yang L., Hornsby P. J. (1998) CYP21 pseudogene transcripts are much less abundant than those from the active gene in normal human adrenocortical cells under various conditions in culture, Mol. Cell. Endocrinol. , 13–19 [DOI] [PubMed] [Google Scholar]
  • 9.Fujii G. H., Morimoto A. M., Berson A. E., Bolen J. B. (1999) Transcriptional analysis of the PTEN/MMAC1 pseudogene, psiPTEN, Oncogene , 1765–1769 [DOI] [PubMed] [Google Scholar]
  • 10.Korneev S. A., Park J. H., O'Shea M. (1999) Neuronal expression of neural nitric oxide synthase (nNOS) protein is suppressed by an antisense RNA transcribed from an NOS pseudogene. J. Neurosci. , 7711–7720 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Tam O. H., Aravin A. A., Stein P., Girard A., Murchison E. P., Cheloufi S., Hodges E., Anger M., Sachidanandam R., Schultz R. M., Hannon G. J. (2008) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature , 534–538 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Brand-Arpon V., Rouquier S., Massa H., de Jong P. J., Ferraz C., Ioannou P. A., Demaille J. G., Trask B. J., Giorgi D. (1999) A genomic region encompassing a cluster of olfactory receptor genes and a myosin light chain kinase (MYLK) gene is duplicated on human chromosome regions 3q13–q21 and 3p13. Genomics , 98–110 [DOI] [PubMed] [Google Scholar]
  • 13.Potier M. C., Chelot E., Pekarsky Y., Gardiner K., Rossier J., Turnell W. G. (1995) The human myosin light chain kinase (MLCK) from hippocampus: cloning, sequencing, expression, and localization to 3qcen-q21. Genomics , 562–570 [DOI] [PubMed] [Google Scholar]
  • 14.Lazar V., Garcia J. G. (1999) A single human myosin light chain kinase gene (MLCK; MYLK). Genomics , 256–267 [DOI] [PubMed] [Google Scholar]
  • 15.Han Y. J., Hu W. Y., Chernaya O., Antic N., Gu L., Gupta M., Piano M., de Lanerolle P. (2006) Increased myosin light chain kinase expression in hypertension: Regulation by serum response factor via an insertion mutation in the promoter. Mol. Biol. Cell , 4039–4050 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.De Lanerolle P., Paul R. J. (1991) Myosin phosphorylation/dephosphorylation and regulation of airway smooth muscle contractility. Am. J. Physiol. Lung Cell. Mol. Physiol. , L1–L14 [DOI] [PubMed] [Google Scholar]
  • 17.Klemke R. L., Cai S., Giannini A. L., Gallagher P. J., de Lanerolle P., Cheresh D. A. (1997) Regulation of cell motility by mitogen-activated protein kinase. J. Cell. Biol. , 481–492 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Yamakita Y., Yamashiro S., Matsumura F. (1994) In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells. J. Cell Biol. , 129–137 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Fishkind D. J., Cao L. G., Wang Y. L. (1991) Microinjection of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytokinesis. J. Cell Biol. , 967–975 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Fazal F., Gu L., Ihnatovych I., Han Y., Hu W., Antic N., Carreira F., Blomquist J. F., Hope T. J., Ucker D. S., de Lanerolle P. (2005) Inhibiting myosin light chain kinase induces apoptosis in vitro and in vivo. Mol. Cell. Biol. , 6259–6266 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Han Y. J., de Lanerolle P. (2008) Naturally extended CT. AG repeats increase H-DNA structures and promoter activity in the smooth muscle myosin light chain kinase gene Mol. Cell. Biol. , 863–872 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Han Y. J., Hu W. Y., Piano M., de Lanerolle P. (2008) Regulation of myosin light chain kinase expression by angiotensin II in hypertension. Am. J. Hypertens. , 860–865 [DOI] [PubMed] [Google Scholar]
  • 23.Dudek S. M., Garcia J. G. (2001) Cytoskeletal regulation of pulmonary vascular permeability. J. Appl. Physiol. , 1487–1500 [DOI] [PubMed] [Google Scholar]
  • 24.Minamiya Y., Nakagawa T., Saito H., Matsuzaki I., Taguchi K., Ito M., Ogawa J. (2005) Increased expression of myosin light chain kinase mRNA is related to metastasis in non-small cell lung cancer. Tumour Biol. , 153–157 [DOI] [PubMed] [Google Scholar]
  • 25.Gallagher P. J., Herring B. P. (1991) The carboxyl terminus of the smooth muscle myosin light chain kinase is expressed as an independent protein, telokin. J. Biol. Chem. , 23945–23952 [PMC free article] [PubMed] [Google Scholar]
  • 26.Yin F., Hoggatt A. M., Zhou J., Herring B. P. (2006) 130-kDa smooth muscle myosin light chain kinase is transcribed from a CArG-dependent, internal promoter within the mouse mylk gene. Am. J. Physiol. Lung Cell. Mol. Physiol. , C1599–C1609 [DOI] [PubMed] [Google Scholar]
  • 27.Jacq C., Miller J. R., Brownlee G. G. (1977) A pseudogene structure in 5S DNA of Xenopus laevis. Cell , 109–120 [DOI] [PubMed] [Google Scholar]
  • 28.Watanabe T., Totoki Y., Toyoda A., Kaneda M., Kuramochi-Miyagawa S., Obata Y., Chiba H., Kohara Y., Kono T., Nakano T., Surani M. A., Sakaki Y., Sasaki H. (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature , 539–543 [DOI] [PubMed] [Google Scholar]
  • 29.Korneev S., O'Shea M. (2002) Evolution of nitric oxide synthase regulatory genes by DNA inversion, Mol. Biol. Evol. , 1228–1233 [DOI] [PubMed] [Google Scholar]
  • 30.Hirotsune S., Yoshida N., Chen A., Garrett L., Sugiyama F., Takahashi S., Yagami K., Wynshaw-Boris A., Yoshiki A. (2003) An expressed pseudogene regulates the messenger-RNA stability of its homologous coding gene. Nature , 91–96 [DOI] [PubMed] [Google Scholar]
  • 31.Korbel J. O., Kim P. M., Chen X., Urban A. E., Weissman S., Snyder M., Gerstein M. B. (2008) The current excitement about copy-number variation: how it relates to gene duplications and protein families, Curr. Opin. Struct. Biol. , 366–374 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Gerstein M., Zheng D. (2006) The real life of pseudogenes. Sci. Am. , 48–55 [DOI] [PubMed] [Google Scholar]
  • 33.Zakut-Houri R., Oren M., Bienz B., Lavie V., Hazum S., Givol D. (1983) A single gene and a pseudogene for the cellular tumour antigen p53, Nature , 594–597 [DOI] [PubMed] [Google Scholar]
  • 34.Dahia P. L., FitzGerald M. G., Zhang X., Marsh D. J., Zheng Z., Pietsch T., von Deimling A., Haluska F. G., Haber D. A., Eng C. (1998) A highly conserved processed PTEN pseudogene is located on chromosome band 9p21, Oncogene , 2403–2406 [DOI] [PubMed] [Google Scholar]
  • 35.Puget N., Gad S., Perrin-Vidoz L., Sinilnikova O. M., Stoppa-Lyonnet D., Lenoir G. M., Mazoyer S. (2002) Distinct BRCA1 rearrangements involving the BRCA1 pseudogene suggest the existence of a recombination hot spot. Am. J. Hum. Genet. , 858–865 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Click here for additional data file. (26KB, doc)
Click here for additional data file. (8.3MB, tif)
Click here for additional data file. (11.4KB, docx)

Articles from The FASEB Journal are provided here courtesy of The Federation of American Societies for Experimental Biology

RESOURCES