Recently, more than 1000 large intergenic noncoding RNAs (lincRNAs) have been reported. These RNAs are evolutionarily conserved in mammalian genomes and thus presumably function in diverse biological processes. Here, we report the identification of lincRNAs that are regulated by p53. One of these lincRNAs (lincRNA-p21) serves as a repressor in p53-dependent transcriptional responses. Inhibition of lincRNA-p21 affects the expression of hundreds of gene targets enriched for genes normally repressed by p53. The observed transcriptional repression by lincRNA-p21 is mediated through the physical association with hnRNP-K. This interaction is required for proper genomic localization of hnRNP-K at repressed genes and regulation of p53 mediates apoptosis. We propose a model whereby transcription factors activate lincRNAs that serve as key repressors by physically associating with repressive complexes and modulate their localization to sets of previously active genes.

A Large Intergenic Noncoding RNA Induced by p53 Mediates Global Gene Repression in the p53 Response

RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs). However, RBMs can be buried within their local RNA structures, thus inhibiting RNA-protein interactions. N(6)-methyladenosine (m(6)A), the most abundant and dynamic internal modification in eukaryotic messenger RNA, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs, but how m(6)A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m(6)A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m(6)A-switch'. We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m(6)A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m(6)A-switches among HNRNPC-binding sites; and global m(6)A reduction decreased HNRNPC binding at 2,798 high-confidence m(6)A-switches. We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.

/pdf/n-6-methyladenosine-dependent-rna-structural-switches-2tnit20rdh.pdf

N 6 -methyladenosine-dependent RNA structural switches regulate RNA–protein interactions

Noncoding RNAs (ncRNAs) constitute the majority of the human transcribed genome. This largest class of RNA transcripts plays diverse roles in a multitude of cellular processes, and has been implicated in many pathological conditions, especially cancer. The different subclasses of ncRNAs include microRNAs, a class of short ncRNAs; and a variety of long ncRNAs (lncRNAs), such as lincRNAs, antisense RNAs, pseudogenes, and circular RNAs. Many studies have demonstrated the involvement of these ncRNAs in competitive regulatory interactions, known as competing endogenous RNA (ceRNA) networks, whereby lncRNAs can act as microRNA decoys to modulate gene expression. These interactions are often interconnected, thus aberrant expression of any network component could derail the complex regulatory circuitry, culminating in cancer development and progression. Recent integrative analyses have provided evidence that new computational platforms and experimental approaches can be harnessed together to distinguish key ceRNA interactions in specific cancers, which could facilitate the identification of robust biomarkers and therapeutic targets, and hence, more effective cancer therapies and better patient outcome and survival.

Noncoding RNA:RNA Regulatory Networks in Cancer.

The investigators, recognizing that coronary artery disease (CAD), including acute myocardial infarction (MI), is the leading cause of death worldwide, carried out a genome-wide associational study on Icelandic patients with MI. Initially more than 300,000 single nucleotide polymorphisms (SNPs) were examined for any association with MI. Ultimately, the study enrolled 4587 cases and 12,769 controls. The strongest association with MI was noted with 3 correlated SNPs, all located within a 190-kb linkage disequilibrium block on chromosome 9p21. The association with MI was replicated in case-control sample sets of persons of European descent from 3 US cities. The identified variant was adjacent to the tumor suppressor genes CDKN2A and CDKN2B. It was associated with MI at a high level of significance (P = 1.2 × 10−20). The proteins encoded by these genes have a key role in regulating cell proliferation, cell senescence, and apoptosis—all important features of atherogenesis.
Approximately 21% of individuals in the study population are homozygous for the variant, and it is estimated that their risk of having MI is 1.64-fold greater than for noncarriers. The risk is 2.02-fold greater for early-onset cases. The population-attributable risk was estimated as 21% for MI in general and 31% for early-onset cases. This is the first common variant found to consistently confer a substantial risk of MI, defined as an odds ratio exceeding 1.20, in multiple case-control groups of European descent. The variant poses a substantial risk from a public health viewpoint, but it explains only a small part of familial clustering of the disease and would not yield high linkage scores. The implication is that other susceptibility variants are as yet unidentified. In addition to MI, there is reason to believe that the present variant might increase the risk of CAD in general. Just how genetic variants influence the pathogenesis of MI remains to be clarified.

A common variant on chromosome 9p21 affects the risk of myocardial infarction

Colorectal cancer (CRC) is the third most common cancer and has a high metastasis and reoccurrence rate. Long noncoding RNAs (lncRNAs) play an important role in CRC growth and metastasis. Recent studies revealed that lncRNAs participate in CRC progression by coordinating with microRNAs (miRNAs) and protein-coding mRNAs. LncRNAs function as competitive endogenous RNAs (ceRNAs) by competitively occupying the shared binding sequences of miRNAs, thus sequestering the miRNAs and changing the expression of their downstream target genes. Such ceRNA networks formed by lncRNA/miRNA/mRNA interactions have been found in a broad spectrum of biological processes in CRC, including liver metastasis, epithelial to mesenchymal transition (EMT), inflammation formation, and chemo-/radioresistance. In this review, we summarize typical paradigms of lncRNA-associated ceRNA networks, which are involved in the underlying molecular mechanisms of CRC initiation and progression. We comprehensively discuss the competitive crosstalk among RNA transcripts and the novel targets for CRC prognosis and therapy.

https://www.mdpi.com/1422-0067/20/22/5758/pdf

Long Noncoding RNA (lncRNA)-Mediated Competing Endogenous RNA Networks Provide Novel Potential Biomarkers and Therapeutic Targets for Colorectal Cancer

Antisense Noncoding RNA in the INK4 Locus (ANRIL) is the prime candidate gene at Chr9p21, the well-defined genetic risk locus associated with multiple human diseases including coronary artery disease (CAD), while little is known regarding its role in the pathological processes. Endothelial dysfunction triggers atherosclerotic processes that are causatively linked to CAD. To evaluate the function of ANRIL in human endothelial cells (ECs), we examined ANRIL expression under pathological stimuli and found ANRIL was markedly induced by pro-inflammatory factors. Loss-of-function and chromatin immunoprecipitation approaches revealed that NF-κB mediates TNF-α induced ANRIL expression. RNA sequencing revealed that ANRIL silencing dysregulated expression of inflammatory genes including IL6 and IL8 under TNF-α treatment. We explored the regulatory mechanism of ANRIL on IL6/8 and found that Yin Yang 1 (YY1), an ANRIL binding transcriptional factor revealed by RNA immunoprecipitation, was required for IL6/8 expression under TNF-α treatment. YY1 was enriched at promoter loci of IL6/8 and ANRIL silencing impaired the enrichment, indicating a cooperation between ANRIL and YY1 in the regulation of inflammatory genes. For the first time, we establish the connection between ANRIL and NF-κB pathway and show that ANRIL regulates inflammatory responses through binding with YY1. The newly identified TNF-α-NF-κB-ANRIL/YY1-IL6/8 pathway enhances understanding of the etiology of CAD and provides potential therapeutic target for treatment of CAD.

Long non-coding RNA ANRIL regulates inflammatory responses as a novel component of NF-κB pathway.

Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) is an example of a functional long noncoding RNA involved in many biologic processes However, the mechanisms for Malat1 in myogenesis are unclear Serum response factor (SRF) is a pivotal transcription factor for muscle proliferation and differentiation and is reported to be a target gene for muscle-specific microRNA-133 (miR-133) In this study, we initially found that silencing Malat1 in the mouse myoblast C2C12 cell line inhibited myocyte differentiation and decreased Srf at both the RNA and protein levels Srf silencing decreased Malat1 expression as well Further study revealed that Malat1 contained an miR-133 functional target site, and the interplay between Malat1 and Srf was miR-133 dependent We demonstrated that Malat1 modulates Srf through miR-133 as a competing endogenous RNA and established a novel connection among Malat1, miR-133, and Srf in myoblast differentiation

Malat1 regulates serum response factor through miR-133 as a competing endogenous RNA in myogenesis.

MALAT-1 interacts with hnRNP C in cell cycle regulation

Heart failure (HF) is associated with mitochondrial dysfunction and energy metabolism impairment MicroRNAs are implicated in the development of heart failure However, the mitochondria enriched microRNA during heart failure remains elusive Here, we generated a pressure overload-induced early and late stage heart failure model at 4 weeks and 8 weeks following transverse aortic constriction (TAC) in mice We found that expression of mitochondrion protein COX4 was highly enriched in isolated mitochondria from cardiac tissues while GAPDH could hardly be detected Furthermore, small RNA sequencing for mitochondria RNAs from failing hearts was performed It was found that 69 microRNAs were upregulated and 2 were downregulated in early heart failure, while 16 microRNAs were upregulated and 6 were downregulated in late heart failure 15 microRNA candidates were measured in both mitochondria and total cardiac tissues of heart failure by real-time PCR MiR-696, miR-532, miR-690, and miR-345-3p were enriched in mitochondria from the failing heart at early stage Bioinformatics analysis showed that mitochondria enriched microRNAs in HF were associated with energy metabolism and oxidative stress pathway For the first time, we demonstrated microRNAs were enriched in mitochondria during heart failure, which established a link between microRNA and mitochondrion in heart failure

/pdf/mitochondria-associated-microrna-expression-profiling-of-3ut296jg0w.pdf

Mitochondria Associated MicroRNA Expression Profiling of Heart Failure

A hybrid cationic bicellar nanodisc was prepared by a conventional Bangham method in combination with a sol–gel reaction and self-assembly process. The relatively small size and disc-like shape was very stable because the incorporation of the organic–inorganic hybrid lipid had formed a crosslinked siloxane net structure on the surface. Physicochemical properties, biocompatibility, intracellular trafficking behavior, in vitro transfection efficiency and in vivo distribution of the bicellar nanodiscs were investigated. The unique characteristics of the cationic PEGylated hybrid bicellar nanodiscs address many of the deficiencies relating to current liposome technology. By optimizing the doping ratio of PEG-containing lipid, we found that a doping ratio of 1 mol% is enough to confer an excellent in vivo delivery performance while not compromising the transfection efficacy in vitro. The distinct disc-like shape, high stability conferred by the hybrid lipid and modest siRNA delivery performance make this platform promising as a siRNA vehicle for efficient siRNA delivery both in vitro and in vivo.

Xiaorui Han

Papers

Long non-coding RNA ANRIL regulates inflammatory responses as a novel component of NF-κB pathway.

Malat1 regulates serum response factor through miR-133 as a competing endogenous RNA in myogenesis.

MALAT-1 interacts with hnRNP C in cell cycle regulation

Mitochondria Associated MicroRNA Expression Profiling of Heart Failure

PEGylated cationic hybrid bicellar nanodisc for efficient siRNA delivery