Expression and function of lncRNA MALAT-1 in the embryonic development of zebrafish

doi:10.1016/j.gene.2018.09.037

Gene

Volume 680, 5 January 2019, Pages 65-71

https://doi.org/10.1016/j.gene.2018.09.037 Get rights and content

Highlights

•
MALAT-1 is mainly expressed in the embryonic nervous system.
•
MALAT-1 highly expressed in the brain, eyes, and heart of zebrafish.
•
MALAT-1 expression is integrant for zebrafish embryonic development.
•
MALAT-1 may be involved in the development of zebrafish brain and heart.

Abstract

Long non-coding RNAs (lncRNAs) are RNA molecules with a transcript length of >200 nt which do not encode proteins, however, they have important functions in the form of RNA at transcriptional level, post-transcriptional level, and epigenetics. MALAT-1 is the first discovered lncRNA in non-small cell lung cancer and its orthologs have been found in zebrafish in the recent years. The present study aimed to determine the expression and possible function of MALAT-1 in the embryonic development of zebrafish. The results of qPCR or in situ hybridization showed that MALAT-1 was dynamically expressed in zebrafish embryonic development and it was widely expressed in brain, eye, heart, and muscle of adult fish. By morpholino knockdown of MALAT-1, zebrafish embryos were found to have smaller body curvature, smaller eyes, enlarged pericardium, and reduced pigmentation. In particular, after morpholino knockdown of MALAT-1, the morphologically defective otic capsule was found to be smaller than the control fish. These results suggest that MALAT-1 may play a key role in the embryonic nervous system development of zebrafish. In addition, we also determined the expressions of seven development-related genes (Scyl1, egr1, oc90, vsx1, nkx2.5, vmhc, and gata4) after MALAT-1 knockdown and found that four genes egr1, nkx2.5, gata4, and vmhc were up-regulated, suggesting a potential regulatory role of MALAT-1 on these development-related genes.

Introduction

In the recent years, with the development of genomics, bioinformatics, and the massive application of high-throughput sequencing technology, it has been found that only <2% of the transcripts in human genome have protein coding function, most of which are non-coding RNA (ncRNA) such as tRNA, rRNA, miRNA, and lncRNA (Adams et al., 2017). Now ncRNA have attracted more and more attention due to their functions in the gene expression regulation. Long non-coding RNAs (lncRNAs) are the ncRNA molecules with a transcript length of >200 nt and transcribed by RNA polymerase II (Ponting and Belgard, 2010). According to the different ways of transcription and localization of lncRNAs, they are roughly divided into the following five types: intergenic lncRNA, bidirectional lncRNA, intron lncRNA, sense lncRNA, and antisense lncRNA (Ponting et al., 2009).

Accumulated studies have shown that lncRNAs have been implicated in numerous biological processes in organisms including cell proliferation, differentiation, metabolism, and apoptosis (Hung et al., 2011), embryonic development (Chambers et al., 2003), cancer (Luo et al., 2013), and other diseases (Huang et al., 2013). They can regulate target genes at the transcriptional, post-transcriptional, and epigenetic levels, respectively (Gutschner and Diederichs, 2012). The regulation of all physiological or pathological processes is closely related, so the elucidation of the function and regulation mechanism of lncRNA is of great significance to modern life sciences.

Embryonic development is a complex process which is regulated by many transcriptional regulatory factors. With the development of high-throughput deep sequencing technology, it has been found that lncRNA plays a very important role in the regulation of animal embryonic development. Dinger et al. (2008) found two developmentally related lncRNAs, Evx1as and HOXB5/6AS, in mouse ES cells. Their CHIP experiments showed that the two LncRNAs were involved in histone methyltransferase and trimethylated H3K4 histones, indicating that lncRNA has a regulatory role in the development and differentiation of ES cells. H19 is the earliest discovered lncRNA and highly expressed in the embryonic stage of mice. With the birth of mice, the expression level of H19 was significantly reduced, but the gene maintained a strong expression signal in mouse skeletal muscle before and after birth. Therefore it may play a crucial role in the development of skeletal muscles. Further studies have demonstrated that LncRNA-H19 encodes miR-675-3p and miR-675-5p, which directly interacts with the transcription factor SMAD (Drosophila mothers against decapentaplegic protein), thereby regulating bone development (Steck et al., 2012). Interestingly, a recent study in zebrafish identified 29 evolutionarily conserved lncRNAs, of which two lncRNAs exhibited a significant regulatory function during zebrafish development as demonstrated by morpholino intervention (Ulitsky et al., 2011).

The metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), also known as nuclear-enriched autosomal transcript 2 (NEAT2), is an important member of the lncRNA family. It was originally discovered in the study of non-small cell lung cancer (NSCLC) in 2003 (Ji et al., 2003). MALAT-1 is specifically localized in the nuclear speckle region of the nuclear nucleosome and is transcribed in this region, but its enrichment in this nuclear plaque presupposes that RNA polymerase II-dependent transcription is activated (Clemson et al., 2009). Phylogenetic analysis indicates that MALAT-1 is highly conserved among mammals (Zhang et al., 2012), however, compared with MALAT-1 in zebrafish, it has only similarity at the 3′ end. Previous studies have shown that nuclear-enriched MALAT-1 interacts with serine/arginine (SR) protein, affecting the distribution of splicing factors. Silencing MALAT-1 or over-expressing SR protein can alter the alternative splicing of endogenous pre-mRNA to varying degrees (Tripathi et al., 2010). The study also found that MALAT-1 regulated the phosphorylation and dephosphorylation of SR proteins at the cellular level, thus controlling their activity. In addition, MALAT-1 also can bind to demethylated Polycomb 2 to promote the relocation and expression of growth genes (Bernstein et al., 2006). Moreover, MALAT-1 has shown to regulate the expressions of cell cycle related genes. B-MYB (Myb-related protein B) is an oncogenic transcription factor involved in the progression of G2/M phase, in the cells silenced by MALAT-1, the splicing factor binding site on the precursor mRNA of B-MYB changes and abnormal splicing occurs (Tripathi et al., 2013). Additionally, MALAT-1 is highly expressed in various tumors such as colorectal cancer (Yang et al., 2015), liver cancer (Wang et al., 2014), and breast cancer (Zhao et al., 2014), and it can promote tumor cells proliferation, metastasis, and invasion.

Currently, there have been only few reports on the function of MALAT-1 in the embryonic development of zebrafish. In the present study, we first determined the expression of MALAT-1 in the embryonic development of zebrafish by quantitative realtime PCR (qPCR) and in situ hybridization. To evaluate the possible function of MALAT-1, specific morpholino was designed to knock-down MALAT-1 and then to determine its effect on the embryonic development. This study provides the clue for further exploration of the role of MALAT-1 in embryonic development.

Section snippets

Experimental animals

Zebrafish used in the experiment was provided by the Animal Center of Qixiu Campus of Nantong University, China. The rearing water was tap water filtered by activated carbon. The oxygen saturation is >80% and the temperature is 28 ± 1 °C. The domesticated photoperiod is 14:10 h of day: night and the illumination is 1000 lx. Spawning was stimulated by turning on the light and the healthy fertilized eggs/embryos were collected under stereomicroscope at 2, 6, 10, 12, 24, 36, 48, and 72 h after

Bioinformatics analysis of zebrafish MALAT-1

We performed the bioinformatics analysis of zebrafish MALAT-1 according to the databases Ensembl genome browser 90 (http://asia.ensembl.org/index.html), lncrnadb (http://www.lncrnadb.org/), and UCSC genome browser (http://genome.ucsc.edu/), and the literature (Ulitsky et al., 2011). We found that MALAT-1 is a ~7.5 kb transcript located on the zebrafish chromosome 14qA (human chromosome 11qA13.1 and mouse chromosome 19qA) with one exon. MALAT-1 orthologs appear in syntenic genomic positions near

Discussion

LncRNA MALAT-1 has been reported to be expressed in a variety of human organs, where it relatively highly expressed in kidney, brain and heart, and it is up-regulated in a range of cancers (Ji et al., 2003; Yamada et al., 2006; Lin et al., 2007; Guffanti et al., 2009). Moreover, MALAT-1 is highly expressed in the brain of mouse. RNA fluorescence in situ hybridization on adult mouse brain sections showed high expression of nuclear-localized MALAT-1 transcripts in pyramidal neurons of the

Conclusion

In conclusion, our results reveal that lncRNA MALAT-1 is not only widely expressed during the embryonic development of zebrafish but also in various organs of adult fish such as brain, eyes, heart, and muscle. We also find that MALAT-1 is highly expressed in the embryonic or adult nervous system of zebrafish. Moreover, MO knockdown of MALAT-1 is almost lethal and its expression is integrant for zebrafish embryonic development. Down-regulation of MALAT-1 expression had significant effect on the

Acknowledgments

This research was supported by the National Natural Science Foundation of China (Grant No. 31472285) and the Key Subjects of Biology in Henan Province, China.

Conflict of interest

The authors declare that there is no conflict of interest.

References (32)

I. Chambers et al.
Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells
Cell
(2003)
C.M. Clemson et al.
An architectural role for a nuclear noncoding RNA: neat1 RNA is essential for the structure of paraspeckles
Mol. Cell
(2009)
C.D. Kuhn et al.
On-enzyme refolding permits small RNA and tRNA surveillance by the cca-adding enzyme
Cell
(2015)
C.P. Ponting et al.
Evolution and functions of long noncoding RNAs
Cell
(2009)
V. Tripathi et al.
The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation
Mol. Cell
(2010)
I. Ulitsky et al.
Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution
Cell
(2011)
J.Y. Wang et al.
Mutual inhibition between YAP and SRSF1 maintains long non-coding RNA, MALAT1- induced tumourigenesis in liver cancer
Cell. Signal.
(2014)
J.E. Wilusz et al.
3′ end processing of a long nuclear-retained non-coding RNA yields a tRNA-like cytoplasmic RNA
Cell
(2008)
M.H. Yang et al.
Malat1 promotes colorectal cancer cell proliferation/migration/invasion via PRKA kinase anchor protein 9
BBA Mol. Basis Dis.
(2015)
B. Zhang et al.
The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory role in the adult
Cell Rep.
(2012)

X. Zhao et al.

Gene targeting reveals the role of Oc90 as the essential organizer of the otoconial organic matrix

Dev. Biol.

(2007)

Z. Zhao et al.

17 β-estradiol treatment inhibits breast cell proliferation, migration and invasion by decreasing malat-1 RNA level

Biochem. Biophys. Res. Commun.

(2014)

B.D. Adams et al.

Targeting non-coding RNAs in disease

J. Clin. Invest.

(2017)

D. Bernard et al.

A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression

EMBO J.

(2010)

E. Bernstein et al.

Mouse polycomb proteins bind differentially to methylated histone h3 and RNA and are enriched in facultative heterochromatin

Mol. Cell. Biol.

(2006)

C.N. Cheng et al.

Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization

JoVE

(2014)

Cited by (24)

Histological characterization and lncRNA-mRNA integrated profiling analysis of the scales in rainbow trout (Oncorhynchus mykiss) under salinity acclimation
2024, Aquaculture
Long noncoding RNAs (lncRNAs) play a multifaceted role in transcriptional regulation and are important regulators of bone metabolism. Bone metabolism of rainbow trout (Oncorhynchus mykiss) has been previously shown to be affected by salinity change, and the regeneration process of scales would serve as an ideal model for studying the regulatory mechanism underlying this process. Here in this study, the effect of salinity acclimation on histological characteristics of scales during a 21-day regenerating process was determined using paraffin section and HE staining; in addition, the calcium (Ca), phosphorus (P) content and the molar mass ratio of calcium to phosphorus (Ca/P) in the regenerated scales were also detected by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The results showed that only the P content was significantly affected by salinity acclimation. In order to further clarify the regulatory mechanism of the regenerated scales in adaptation to the salinity change, the global lncRNA expression profiles of both the regenerated and ontogenic scales of rainbow trout under salinity acclimation were examined using Illumina sequencing platform. As a result, a total of 612 significantly differentially expressed (DE) lncRNAs were identified in the regenerated scales, and target gene prediction of these DE lncRNAs has obtained 2330 DE target genes (DETGs). GO and KEGG analysis showed that the aforementioned DETGs were significantly annotated to GO terms including S-adenosylmethionine-homocysteine S-methyltransferase activity, cobalamin binding, etc.; and enriched in bone metabolism related signaling pathways such as hypertrophic cardiomyopathy and dilated cardiomyopathy signaling. As for the ontogenic scales, the STEM analysis of 3432 DE lncRNAs showed that eight patterns (consisted of 1093 DE lncRNAs) were significantly clustered (P < 0.05) at different time points during the salinity acclimation (7, 14 and 21d), and a total of 201 DETGs were subsequently obtained. Furthermore, the DE lncRNAs respectively identified from the regenerated and ontogenic scales collected at 14d were compared using the Venn diagram, a total of 81 DE lncRNAs were identified in both scale types, while 531 and 653 DE lncRNAs were respectively found to be specially identified in the regenerated and ontogenic scales. GO and KEGG analysis of the DETGs respectively predicted from the aforementioned DE lncRNAs also revealed significantly different enriched functions and pathways. Co-expression network of these DE lncRNA and DETGs suggested that 66 DE lncRNA and 57 DETGs have generated a network of 77 pairs of relationships in the regenerated scales, while 12 DE lncRNAs and 12 DETGs produced 12 relationship pairs in the ontogenic scales. The results indicated that they might be related to the functions including metal ion binding, membrane and nucleotide binding, etc. The DE lncRNAs and DETGs identified in this study might provide reference information for further research on the regulation of bone metabolism in teleost species.
Maternal NO<inf>2</inf> exposure disturbs the long noncoding RNA expression profile in the lungs of offspring in time-series patterns
2022, Ecotoxicology and Environmental Safety
Citation Excerpt :
The fetal origin of adult disease (FOAD) hypothesis proposes that adverse intrauterine environments, such as air pollution, can lead to adverse birth outcomes in offspring or an increased risk of disease in adulthood (Barker, 1990; Byrne and Phillips, 2000). Importantly, lncRNAs were found to play an important role in regulating embryonic development (Wu et al., 2019). Placental lncRNA Neat1 expression levels positively correlate with intrauterine growth restriction (Paquette et al., 2021).
Gestation is a sensitive window to nitrogen dioxide (NO₂) exposure, which may disturb fetal lung development and lung function later in life. Animal and epidemiological studies indicated that long noncoding RNAs (lncRNAs) participate in abnormal lung development induced by environmental pollutant exposure. In the present study, pregnant C57BL/6J mice were exposed to 2.5 ppm NO₂ (mimicking indoor occupational exposure) or clean air, and lncRNAs expression profiles in the lungs of offspring mice were determined by lncRNA-seq on embryonic day 13.5 (E13.5), E18.5, postnatal day 1 (P1), and P14. The lung histopathology examination of offspring was performed, followed by weighted gene coexpression network analysis (WGCNA), prediction of lncRNAs-target genes, and the biological processes enrichment analysis of lncRNAs. Our results indicated that maternal NO₂ exposure induced hypoalveolarization on P14 and differentially expressed lncRNAs showed a time-series pattern. Following WGCNA and enrichment analysis, 2 modules participated in development-related pathways. Importantly, the expressions of related genes were altered, some of which were confirmed to be related to abnormal vascular development and even lung diseases. The research points out that the maternal NO₂ exposure leads to abnormal lung development in offspring that might be related to altered lncRNAs expression profiles with time-series-pattern.
The potential role of eyestalk in the immunity of Litopenaeus vannamei to Vibrio parahaemolyticus infection II. From the perspective of long non-coding RNA
2022, Fish and Shellfish Immunology
Citation Excerpt :
In tilapia, lncRNAs were found to have an important role in the response to cold, salt, and hypoxia stressors [9]. Furthermore, through transcriptional regulation of targeted genes, lncRNA has been demonstrated to play a significant role in the embryonic development of Cyprinus carpio [12] and the embryonic nervous system of zebrafish [13]. Simultaneously, through altering mRNA expression, lncRNAs have been demonstrated to stimulate muscle cell growth in Haliotis discus hannai [14].
Long non-coding RNAs (lncRNAs) have been linked to immunological modulation. Unfortunately, little is known about the processes of immune control in shrimp. In crustaceans such as Litopenaeus vannamei, a prominent aquaculture species, the X-organ-sinus gland complex (XO-SG) in the eyestalk is an essential neuroendocrine regulatory organ. Eyestalk ablation is commonly employed in aquaculture to accelerate ovarian maturation in shrimp. It does, however, have a negative impact on the shrimps' immunocompetence and causes death. As a result, we used RNA-seq to profile the transcriptomes of L. vannamei hemocytes infected with Vibrio parahaemolyticus after the eyestalk ablation. Following strict transcript screening procedures, 2307 lncRNAs were identified from L. vannamei hemocytes in this study. Pearson correlation analysis was finally used to uncover 535 DElncRNAs and 1566 DEmRNA targets. According to the Venn diagram analysis, 326 non-eyestalk regulatory lncRNAs (NElncRNAs) with a target of 1014 non-eyestalk regulatory genes (NEmRNAs), 47 eyestalk negative regulatory lncRNAs (ENRlncRNAs) with a target of 95 eyestalk negative regulatory genes (ENRmRNAs), and 162 eyestalk positive regulatory lncRNAs (EPRlncRNAs) with a target of 457 eyestalk positive regulatory genes (EPRmRNAs) were screened. The bioinformatics analysis revealed that lncRNAs were associated with Axon regeneration, Rap1 signaling pathway, Thyroid hormone signaling pathway, TGF-beta signaling pathway, and PI3K-Akt signaling pathway, implying that lncRNAs may play a role in the regulation of the neuroendocrine-immune (NEI) system. Furthermore, several lncRNAs targeting HSP70, YWHAZ, FER2, HIF1α, and Notch were discovered and verified by qRT-PCR. These findings showed that regulation of lncRNAs in hemocytes which were controlled by the eyestalk might be one of the impact variables in controlling the differential expression of mRNAs associated with immune response in L. vannamei infected with V. parahaemolyticus.
lncRNA FDNCR promotes apoptosis of granulosa cells by targeting the miR-543-3p/DCN/TGF-β signaling pathway in Hu sheep
2021, Molecular Therapy Nucleic Acids
Citation Excerpt :
Recently, numerous lncRNAs have been identified in humans and other organisms that play diverse roles in regulating various phenomenon, such as cell proliferation and apoptosis,7,8 cell development,9 cell differentiation,10 and development of certain diseases.11,12 Specifically, lncRNAs are involved in regulating spermatogenesis,13,14 steroidogenesis,15 embryo implantation16 and development,17 follicular development,18 oocyte maturation,19 GC apoptosis and proliferation,20,21 and reproductive diseases,22,23 suggesting their importance in reproduction.24 However, only a few lncRNAs have been identified in domestic animals.
Long non-coding RNAs (lncRNAs) regulate the development of follicles and reproductive diseases, but the mechanisms by which lncRNAs regulate ovarian functions and fertility remain elusive. We profiled the expression of lncRNAs in ovarian tissues of Hu sheep with different prolificacy and identified 21,327 lncRNAs. Many of the lncRNAs were differentially expressed in different groups. We further characterized an lncRNA that was predominantly expressed in the ovaries of the low prolificacy Fec^B+ (LPB+) group and mainly present in granulosa cells (GCs), and the expression of this lncRNA decreased during follicular development, which we named follicular development-associated lncRNA (FDNCR). Next, we found that FDNCR directly binds miR-543-3p, and decorin (DCN) was identified as a target of miR-543-3p. FDNCR overexpression promoted GC apoptosis through increased expression of DCN, which could be attenuated by miR-543-3p. Furthermore, miR-543-3p increased and FDNCR reduced the expression of transforming growth factor-β (TGF-β) pathway-related genes, including TGF-β1 and inhibin beta A (INHBA), which were upregulated upon DCN silencing. Our results demonstrated that FDNCR sponges miR-543-3p in GCs and prevents miR-543-3p from binding to the DCN 3′ UTR, resulting in DCN transactivation and TGF-β pathway inhibition and promotion of GC apoptosis in Hu sheep. These findings provide insights into the mechanisms underlying prolificacy in sheep.
The stage-specific long non-coding RNAs and mRNAs identification and analysis during early development of common carp, Cyprinus carpio
2021, Genomics
Citation Excerpt :
In zebrafish, lncrps25 plays an essential role in motor neuron development through controlling the expression of Olig2 [24]. LncRNA MALAT-1 may play a key role in the embryonic nervous system development of zebrafish by regulating development-related genes [25]. We further analyzed the trans-targeted mRNA functions of lncrps25 and lncRNA malat1, and 126 and 231 KEGG pathways were identified respectively.
Cyprinus carpio is considered an alternative vertebrate fish model to zebrafish. However, systemic times-series research on the lncRNAs and mRNAs during early development of C. carpio has not been reported yet. This study provides the first long non-coding RNA (lncRNA)-mRNA expression profiles during six main early development stages (2 h post-fertilization hpf, 6 hpf, 12 hpf, 20 hpf, 64 hpf and 1 day post-hatching). A total of 51,979 lncRNAs were identified. We screened the top 10 abundance lncRNAs and mRNAs and stage-specific lncRNAs and mRNAs (specificity measure SPM > 0.9). We identified significant differentially expressed lncRNAs and mRNAs (|log2 (fold change)| ≥ 1 and false discovery rate FDR of <0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified numerous signaling pathways. Additionally, the lncRNA-mRNA co-regulated network analysis of two lncRNAs (lncrps25 and malat1) and two mRNAs (mitf and troponin T) were investigated. Our results provide new insight into the role of lncRNAs and mRNAs, and would advance the understanding of lncRNA-mediated mechanisms in early development of fish.
Role of epigenetics in zebrafish development
2019, Gene
Citation Excerpt :
In contrast to the role of lncRNAs in development, another recent study by Goudarzi et al. (2018) knocked down the 25 developmentally important lncRNAs, which was shown to be dispensable for embryo development in zebrafish. However, a recent study done by Wu and his colleagues (2019) shows that lncRNA MALAT-1 mutants showed a deficit in the size of the otic capsule when compared to control (Wu et al., 2019). MALAT-1 was found to be a zygotic gene.
The role of epigenetics in development has garnered attention in recent years due to their ability to modulate the embryonic developmental gene expression in response to the environmental cues. The epigenetic mechanisms - DNA methylation, histone modification, and non-coding RNAs have a unique impact on vertebrate development. Zebrafish, a model vertebrate organism is being used widely in developmental studies due to their high fecundability and rapid organogenesis. With increased studies on various aspects of epigenetics in development, this review gives a glimpse of the major epigenetic modifications and their role in zebrafish development. In this review, the basic mechanism behind each modification followed by their status in zebrafish has been reviewed. Further, recent advancements in the epigenetic aspect of zebrafish development have been discussed.

View all citing articles on Scopus

View full text

Research paperExpression and function of lncRNA MALAT-1 in the embryonic development of zebrafish

Highlights

Abstract

Introduction

Section snippets

Experimental animals

Bioinformatics analysis of zebrafish MALAT-1

Discussion

Conclusion

Acknowledgments

Conflict of interest

Cell

Mol. Cell

Cell

Cell

Mol. Cell

Cell

Cell. Signal.

Cell

BBA Mol. Basis Dis.

Cell Rep.

Dev. Biol.

Biochem. Biophys. Res. Commun.

Targeting non-coding RNAs in disease

J. Clin. Invest.

A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression

EMBO J.

Mouse polycomb proteins bind differentially to methylated histone h3 and RNA and are enriched in facultative heterochromatin

Mol. Cell. Biol.

Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization

JoVE

Research paper
Expression and function of lncRNA MALAT-1 in the embryonic development of zebrafish