DGCR8 Microprocessor Subunit Mutation and Expression Deregulation in Thyroid Lesions.

DGCR8 emerged recently as miRNAs biogenesis pathway protein with a highlighted role in thyroid disease. This study aimed to characterize this miRNA biogenesis component, in particular the p.(E518K) mutation and DGCR8 expression in a series of thyroid lesions. The series of thyroid lesions was genotyped for the c.1552G>A p.(E518K) mutation. When frozen tissue was available, DGCR8 mRNA expression was analysed by qPCR. Formalin-fixed paraffin-embedded tissues were studied for DGCR8 immunoexpression. We present for the first time the p.(E518K) mutation in a case of poorly differentiated thyroid carcinoma and present the deregulation of DGCR8 expression at mRNA level in follicular-patterned tumours. The obtained data solidify DGCR8 as another important player of miRNA-related gene mutations in thyroid tumorigenesis, particularly in follicular-patterned thyroid tumours.

Microprocessor-dependent processing of splice site overlapping microRNA exons does not result in changes in alternative splicing.

MicroRNAs are found throughout the genome and are processed by the microprocessor complex (MPC) from longer precursors. Some precursor miRNAs overlap intron:exon junctions. These splice site overlapping microRNAs (SO-miRNAs) are mostly located in coding genes. It has been intimated, in the rarer examples of SO-miRNAs in noncoding RNAs, that the competition between the spliceosome and the MPC modulates alternative splicing. However, the effect of this overlap on coding transcripts is unknown. Unexpectedly, we show that neither Drosha silencing nor SF3b1 silencing changed the inclusion ratio of SO-miRNA exons. Two SO-miRNAs, located in genes that code for basal membrane proteins, are known to inhibit proliferation in primary keratinocytes. These SO-miRNAs were up-regulated during differentiation and the host mRNAs were down-regulated, but again there was no change in inclusion ratio of the SO-miRNA exons. Interestingly, Drosha silencing increased nascent RNA density, on chromatin, downstream from SO-miRNA exons. Overall our data suggest a novel mechanism for regulating gene expression in which MPC-dependent cleavage of SO-miRNA exons could cause premature transcriptional termination of coding genes rather than affecting alternative splicing.

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting.

During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem-loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.

Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

ERH: a plug-and-play protein important for gene silencing and cell cycle progression.

In metazoans, most proteins have pleiotropic cellular functions and have the ability to interact with several factors to accomplish these different functions. This is the case of eukaryotic ERH proteins, a family of homodimeric proteins involved in DNA replication and cell cycle control as well as in gene silencing by contributing either to the biogenesis of small interference RNAs (miRNAs, piRNAs) or to the recruitment of RNA decay machineries. Very recently, several crystal structures describing complexes formed by eukaryotic ERH proteins and several small peptides from various partners have highlighted the existence of different binding sites on the surface of ERH proteins. In this issue of The FEBS Journal, Wang et al. present the crystal structure of the complex formed between the human ERH protein and a short peptide of the CIZ1 protein, one of its partners. Altogether, this information will be particularly important for future studies aimed at dissecting the different biological functions governed by this family of highly conserved proteins. Comment on: https://doi.org/10.1111/febs.16611.

MicroRNAs as Key Effectors in the p53 Network.

The guardian of the genome p53 is embedded in a fine-spun network of MicroRNAs. p53 is able to activate or repress directly the transcription of MicroRNAs that are participating in the tumor-suppressive mission of p53. On the other hand, the expression of p53 is under tight control of MicroRNAs that are either targeting directly p53 or factors that are modifying its protein level or activity. Although the most important function of p53 is suggested to be transcriptional regulation, there are several nontranscriptional functions described. One of those regards the modulation of MicroRNA biogenesis. Wild-type p53 is increasing the maturation of selected MicroRNAs from the primary transcript to the precursor MiRNA by interacting with the Microprocessor complex. Furthermore, p53 is modulating the mRNA accessibility for certain MicroRNAs by association with the RISC complex and transcriptional regulation of RNA-binding proteins. In this way p53 is able to remodel the MiRNA-mRNA interaction network. As wild-type p53 is employing MicroRNAs to suppress cancer development, gain-of-function mutant p53 proteins use MicroRNAs to confer oncogenic properties like chemoresistance and the ability to drive metastasis. Like its wild-type counterpart mutant p53 is able to regulate MicroRNAs transcriptionally and posttranscriptionally. Mutant p53 affects the MiRNA processing at two cleavage steps through interfering with the Microprocessor complex and by downregulating Dicer and KSRP, a modulator of MiRNA biogenesis. Thus, MicroRNAs are essential components in the p53 pathway, contributing substantially to combat or enhance tumor development depending on the wild-type or mutant p53 context.

Dysregulation of microRNA biogenesis in cancer: the impact of mutant p53 on Drosha complex activity.

A widespread decrease of mature microRNAs is often observed in human malignancies giving them potential to act as tumor suppressors. Thus, microRNAs may be potential targets for cancer therapy. The global miRNA deregulation is often the result of defects in the miRNA biogenesis pathway, such as genomic mutation or aberrant expression/localization of enzymes and cofactors responsible of miRNA maturation. Alterations in the miRNA biogenesis machinery impact on the establishment and development of cancer programs. Accumulation of pri-microRNAs and corresponding depletion of mature microRNAs occurs in human cancers compared to normal tissues, strongly indicating an impairment of crucial steps in microRNA biogenesis. In agreement, inhibition of microRNA biogenesis, by depletion of Dicer1 and Drosha, tends to enhance tumorigenesis in vivo. The p53 tumor suppressor gene, TP53, is mutated in half of human tumors resulting in an oncogene with Gain-Of-Function activities. In this review we discuss recent studies that have underlined a role of mutant p53 (mutp53) on the global regulation of miRNA biogenesis in cancer. In particular we describe how a new transcriptionally independent function of mutant p53 in miRNA maturation, through a mechanism by which this oncogene is able to interfere with the Drosha processing machinery, generally inhibits miRNA processing in cancer and consequently impacts on carcinogenesis.