Regulation of pri-miRNA processing by a long noncoding RNA transcribed from an ultraconserved region.

Noncoding RNAs (ncRNAs) control cellular programs by affecting protein-coding genes, but evidence increasingly points to their involvement in a network of ncRNA-ncRNA interactions. Here, we show that a long ncRNA, Uc.283+A, controls pri-miRNA processing. Regulation requires complementarity between the lower stem region of the pri-miR-195 transcript and an ultraconserved sequence in Uc.283+A, which prevents pri-miRNA cleavage by Drosha. Mutation of the site in either RNA molecule uncouples regulation in vivo and in vitro. We propose a model in which lower-stem strand invasion by Uc.283+A impairs microprocessor recognition and efficient pri-miRNA cropping. In addition to identifying a case of RNA-directed regulation of miRNA biogenesis, our study reveals regulatory networks involving different ncRNA classes of importance in cancer.

Microprocessor dynamics shows co- and post-transcriptional processing of pri-miRNAs.

miRNAs are small regulatory RNAs involved in the regulation of translation of target transcripts. miRNA biogenesis is a multistep process starting with the cleavage of the primary miRNA transcript in the nucleus by the Microprocessor complex. Endogenous processing of pri-miRNAs is challenging to study and the in vivo kinetics of this process is not known. Here, we present a method for determining the processing kinetics of pri-miRNAs within intact cells over time, using a pulse-chase approach to label transcribed RNA during 15 min, and follow the processing within a 1-hour window after labeling with bromouridine. We show that pri-miRNAs exhibit different processing kinetics ranging from fast over intermediate to slow processing, and we provide evidence that pri-miRNA processing can occur both cotranscriptionally and post-transcriptionally.

lncRNAs and microRNAs with a role in cancer development.

Most diseases, including human cancer, are frequently associated with an altered transcription pattern. The alteration of the transcriptome is not restricted to the production of aberrant levels of protein-coding RNAs, but also refers to the dysregulation of the expression of the multiple noncoding members that comprise the human genome. Unexpectedly, recent RNA-seq data of the human transcriptome have revealed that less than 2% of the genome encodes protein-coding transcripts, even though the vast majority of the genome is actively transcribed into non-coding RNAs (ncRNAs) under different conditions. In this review, we present an updated version of the mechanistic aspects of some long non-coding RNAs (lncRNAs) that play critical roles in human cancer. Most importantly, we focus on the interplay between lncRNAs and microRNAs, and the importance of such interactions during the tumorigenic process, providing new insight into the regulatory mechanisms underlying several ncRNA classes of importance in cancer, particularly transcribed ultraconserved regions (T-UCRs). This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

Long ncRNA A-ROD activates its target gene DKK1 at its release from chromatin.

Long ncRNAs are often enriched in the nucleus and at chromatin, but whether their dissociation from chromatin is important for their role in transcription regulation is unclear. Here, we group long ncRNAs using epigenetic marks, expression and strength of chromosomal interactions; we find that long ncRNAs transcribed from loci engaged in strong long-range chromosomal interactions are less abundant at chromatin, suggesting the release from chromatin as a crucial functional aspect of long ncRNAs in transcription regulation of their target genes. To gain mechanistic insight into this, we functionally validate the long ncRNA A-ROD, which enhances DKK1 transcription via its nascent spliced released form. Our data provide evidence that the regulatory interaction requires dissociation of A-ROD from chromatin, with target specificity ensured within the pre-established chromosomal proximity. We propose that the post-transcriptional release of a subset of long ncRNAs from the chromatin-associated template plays an important role in their function as transcription regulators.

Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR.

Metastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors.