Outlook

Molecular evolutionary evidence that H19 mRNA is functional

KaKs_Calculator 3.0 is an updated toolkit that is capable of calculating selective pressure on both coding and non-coding sequences. Similar to the nonsynonymous/synonymous substitution rate ratio for coding sequences, selection on non-coding sequences can be quantified as the ratio of non-coding nucleotide substitution rate to synonymous substitution rate of adjacent coding sequences. As testified on empirical data, KaKs_Calculator 3.0 shows effectiveness to detect the strength and mode of selection operated on molecular sequences, accordingly demonstrating its great potential to achieve genome-wide scan of natural selection on diverse sequences and identification of potentially functional elements at a whole-genome scale. The package of KaKs_Calculator 3.0 is freely available for academic use only at https://ngdc.cncb.ac.cn/biocode/tools/BT000001.

The long non-coding RNAs (lncRNAs) have gained much attention due to its essential roles in molecular regulation. As one of the classic lncRNAs, H19 is strongly expressed during embryogenesis and plays a crucial biological function during development. Mesenchymal stem cells (MSCs) are an ideal cell source for tissue engineering in musculoskeletal system as they own the multi-differentiation ability towards osteogenesis, adipogenesis, tenogenesis or chondrogenesis. In recent years, many studies have been found in the field of H19 mediated cellular differentiation of MSCs. Here, we summarized the current understanding of H19 during multi-differentiation of MSCs and its application in tissue regeneration of musculoskeletal system. Particularly, its molecular regulation and biological function during the multi-differentiation were also discussed.

H19 was one of the earliest identified, and is the most studied, long noncoding RNAs. It is presumed that H19 is essential for regulating development and disease conditions, and it is associated with carcinogenesis for many types. However the biological function and regulatory mechanism of this conserved RNA, particularly with respect to its effect on transcription, remain largely unknown.

We performed RNA pulldown, RNA immunoprecipitation and deletion mapping to identify the proteins that are associated with H19. In addition, we employed EU (5-ethynyl uridine) incorporation, immunoprecipitation and Western blotting to investigate the functional aspects of H19.

Our research further verifies that H19 is bound to hnRNP U, and this interaction is located within the 5′ 882 nt region of H19. Moreover, H19 disrupts the interaction between hnRNP U and actin, which inhibits phosphorylation at Ser5 of the RNA polymerase II (Pol II) C-terminal domain (CTD), consequently preventing RNA Pol II-mediated transcription. We also showed that hnRNP U is essential for H19-mediated transcription repression.

In this study, we demonstrate that H19 inhibits RNA Pol II-mediated transcription by disrupting the hnRNP U–actin complex.

These data suggest that H19 regulates general transcription and exerts wide-ranging effects in organisms.

miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.

To characterize the H19 gene imprinting in the placental tissues of normal pregnancy and pre-eclampsia.

A total of 188 subjects at 5–9 weeks, 54 at 10–12 weeks, 50 at the second trimester, 65 at term of gestation, and 30 with pre-eclampsia at the third trimester were recruited. Their DNA and RNA were extracted from the placental tissues. The H19 genotypes for individuals were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the H19 gene imprinting was identified by reverse transcription-PCR (RT-PCR) and the RsaI-based RFLP. The levels of H19 RNA transcripts in the pre-eclampsia patients with the H19 heterozygote were measured by quantitative RT-PCR. The frequency of the H19 heterozygote, the H19 imprinting status among different trimesters of pregnancy and the clinical symptoms in the pre-eclampsia patients were analyzed.

(1) About 40–46% of subjects at varying stages of normal pregnancy showed the H19 heterozygote, and 24 out of 87 (27.59%) heterozygous cases were at 5–9 weeks of gestation, but no single one in other stages displayed biallelic expression of the H19 gene. (2) Thirteen out of 30 pre-eclampsia patients were H19 heterozygous and six of these showed the biallelic expression of the H19 gene (loss of imprinting). (3) The pre-eclampsia patients with the biallelic expression of H19 tended to have severer hypertension although their H19 RNA transcription level was similar to that in those with monoallelic expression.

(1) The biallelic expression of the H19 gene exists in some cases at the early stage of normal pregnancy and changes into monoallelic expression near 10 weeks of gestation. The dynamic alternations in the patterns of the H19 gene imprinting may regulate the maintenance of normal pregnancy. (2) The loss of the H19 gene imprinting in the placental tissues of pre-eclampsia patients may be associated with severe hypertension, contributing to the pathogenic process of pre-eclampsia.

IMP-3, a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein (IMP) family, is expressed mainly during embryonic development and in some tumors. Thus, IMP-3 is considered to be an oncofetal protein. The functional significance of IMP-3 is not clear. To identify the functions of IMP-3 in target gene expression and cell proliferation, RNA interference was employed to knock down IMP-3 expression. Using human K562 leukemia cells as a model, we show that IMP-3 protein associates with IGF-II leader-3 and leader-4 mRNAs and H19 RNA but not c-myc and β-actin mRNAs in vivo by messenger ribonucleoprotein immunoprecipitation analyses. IMP-3 knock down significantly decreased levels of intracellular and secreted IGF-II without affecting IGF-II leader-3, leader-4, c-myc, or β-actin mRNA levels and H19 RNA levels compared with the negative control siRNA treatment. Moreover, IMP-3 knock down specifically suppressed translation of chimeric IGF-II leader-3/luciferase mRNA without altering reporter mRNA levels. Together, these results suggest that IMP-3 knock down reduced IGF-II expression by inhibiting translation of IGF-II mRNA. IMP-3 knock down also markedly inhibited cell proliferation. The addition of recombinant human IGF-II peptide to these cells restored cell proliferation rates to normal. IMP-3 and IMP-1, two members of the IMP family with significant structural similarity, appear to have some distinct RNA targets and functions in K562 cells. Thus, we have identified IMP-3 as a translational activator of IGF-II leader-3 mRNA. IMP-3 plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells.