Proteinase-activated Receptor 2 Promotes Cancer Cell Migration through RNA Methylation-mediated Repression of miR-125b

Proteinase activated-receptor 2 (PAR₂) participates in cancer metastasis promoted by serine proteinases. The current study aimed to test the molecular mechanism by which PAR₂ promotes cancer cell migration. In different cancer cells, activation of PAR₂ by activating peptide (PAR₂-AP) dramatically increased cell migration, whereas knock down of PAR₂ inhibited cellular motility. The PAR₂ activation also repressed miR-125b expression while miR-125b mimic successfully blocked PAR₂-induced cell migration. Moreover, Grb associated-binding protein 2 (Gab2) was identified as a novel target gene of miR-125b and it mediated PAR₂-induced cell migration. The correlation of PAR₂ with miR-125b and Gab2 was further supported by the findings obtained from human colorectal carcinoma specimens. Remarkably, knock down of NOP2/Sun domain family, member 2 (NSun2), a RNA methyltransferase, blocked the reduction in miR-125b induced by PAR₂. Furthermore, PAR₂ activation increased the level of N⁶-methyladenosine (m⁶A)-containing pre-miR-125b in NSun2-dependent manner. Taken together, our results demonstrated that miR-125b mediates PAR₂-induced cancer cell migration by targeting Gab2 and that NSun2-dependent RNA methylation contributes to the down-regulation of miR-125b by PAR₂ signaling. These findings suggest a novel epigenetic mechanism by which microenvironment regulates cancer cell migration by altering miRNA expression.     

NSun2 promotes cell migration through methylating autotaxin mRNA

NSun2 is an RNA methyltransferase introducing 5-methylcytosine into tRNAs, mRNAs, and noncoding RNAs, thereby influencing the levels or function of these RNAs. Autotaxin (ATX) is a secreted glycoprotein and is recognized as a key factor in converting lysophosphatidylcholine into lysophosphatidic acid (LPA). The ATX-LPA axis exerts multiple biological effects in cell survival, migration, proliferation, and differentiation. Here, we show that NSun2 is involved in the regulation of cell migration through methylating ATX mRNA. In the human glioma cell line U87, knockdown of NSun2 decreased ATX protein levels, whereas overexpression of NSun2 elevated ATX protein levels. However, neither overexpression nor knockdown of NSun2 altered ATX mRNA levels. Further studies revealed that NSun2 methylated the 3′-UTR of ATX mRNA at cytosine 2756 in vitro and in vivo. Methylation by NSun2 enhanced ATX mRNA translation. In addition, NSun2-mediated 5-methylcytosine methylation promoted the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. Knockdown of NSun2 suppressed the migration of U87 cells, which was rescued by the addition of LPA. In summary, we identify NSun2-mediated methylation of ATX mRNA as a novel mechanism in the regulation of ATX.     

Functional Study of One Nucleotide Mutation in Pri-MiR-125a Coding Region which Related to Recurrent Pregnancy Loss

MicroRNAs (miRNAs) are short non-coding RNAs which modulate gene expression by binding to complementary segments present in the 3′UTR of the mRNAs of protein coding genes. MiRNAs play very important roles in maintaining normal human body physiology conditions, meanwhile, abnormal miRNA expressions have been found related to many human diseases spanning from psychiatric disorders to malignant cancers. Recently, emerging reports have indicated that disturbed miRNAs expression contributed to the pathogenesis of recurrent pregnancy loss (RPL). In this study, we identified a new mutation site (+29A>G, position relative to pre-miR-125a) by scanning pri-miR-125a coding region in 389 Chinese Han RPL patients. This site was co-existed with two polymorphisms (rs12976445 and rs41275794) in patients heterogeneously and changed the predicted secondary structures of pri-miR-125a. Subsequent in vitro analysis indicated that the A>G mutation reduced mature miR-125a expression, and further led to less efficient inhibition of verified target genes. Functional analysis showed that mutant pri-mir-125a can enhance endometrial stromal cells (ESCs) invasive capacity and increase the sensitivity of ESCs cells to mifepristone. Moreover, we further analyzed the possible molecular mechanism by RIP-chip assay and found that mutant pri-mir-125a disturbed the expression of miR-125a targetome, the functions of which includes embryonic development, cell proliferation, migration and invasion. These data suggest that A>G mutation in pri-miR-125a coding region contributes to the genetic predisposition to RPL by disordering the production of miR-125a, which consequently meddled in gene regulatory network between mir-125a and mRNA.     

NSun2 Promotes Cell Growth via Elevating Cyclin-Dependent Kinase 1 Translation

The tRNA methytransferase NSun2 promotes cell proliferation, but the molecular mechanism has not been elucidated. Here, we report that NSun2 regulates cyclin-dependent kinase 1 (CDK1) expression in a cell cycle-dependent manner. Knockdown of NSun2 decreased the CDK1 protein level, while overexpression of NSun2 elevated it without altering CDK1 mRNA levels. Further studies revealed that NSun2 methylated CDK1 mRNA in vitro and in cells and that methylation by NSun2 enhanced CDK1 translation. Importantly, NSun2-mediated regulation of CDK1 expression had an impact on the cell division cycle. These results provide new insight into the regulation of CDK1 during the cell division cycle.     

Alternative Processing of Primary microRNA Transcripts by Drosha Generates 5′ End Variation of Mature microRNA

Background

It is generally believed that the miRNA processing machinery ensures the generation of a mature miRNA with a fixed sequence, particularly at its 5′ end. However, we and others have recently noted that the ends of a given mature miRNA are not absolutely fixed, but subject to variation. Neither the significance nor the mechanism behind the generation of such miRNA polymorphism is understood. miR-142 is an abundantly expressed miRNA in hematopoietic cells and exhibits a high frequency of 5′ end polymorphism.

Methodology/Principal Findings

Here we show that a shift in the Drosha processing of pri-miRNA generates multiple forms of miR-142s in vivo with differing 5′ ends that might target different genes. Sequence analysis of several pre-miRNA ends cloned from T cells reveals that unlike many other pri-miRNAs that are processed into a single pre-miRNA, pri-miR-142 is processed into 3 distinct pre-miR-142s. Dicer processing studies suggest that each of the 3 pre-miR-142s is processed into a distinct double-stranded miRNA, giving rise to 4 mature miRNA variants that might regulate different target gene pools.

Conclusions/Significance

Thus, alternative Drosha processing might be a novel mechanism for diversification of the miRNA target gene pool.     

序列变异对miR-378生物发生以及靶标关系的影响

本研究旨在探索序列变异对miR-378的结构、表达水平以及靶标关系的影响.利用PCR测序比对不同猪种miR-378的序列突变,预测突变对miR-378二级结构和自由能的改变;构建miR-378表达载体检测突变对其加工过程中各级产物表达水平的作用;运用TargetScan和TargetRank对突变引起的靶标关系变化进行...     

The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a

hnRNP A1 is an RNA-binding protein involved in various aspects of RNA processing. Use of an in vivo cross-linking and immunoprecipitation protocol to find hnRNP A1 RNA targets resulted in the identification of a microRNA (miRNA) precursor, pre-miR-18a. This microRNA is expressed as part of a cluster of intronic RNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92, and potentially acts as an oncogene. Here we show that hnRNP A1 binds specifically to the primary RNA sequence pri-miR-18a before Drosha processing. HeLa cells depleted of hnRNP A1 have reduced in vitro processing activity with pri-miR-18a and also show reduced abundances of endogenous pre-miR-18a. Furthermore, we show that hnRNP A1 is required for miR-18a-mediated repression of a target reporter in vivo. These results underscore a previously uncharacterized role for general RNA-binding proteins as auxiliary factors that facilitate the processing of specific miRNAs.