Calumenin knockdown, by intronic artificial microRNA, to improve expression efficiency of the recombinant human coagulation factor IX.

OBJECTIVES: To improve the expression efficiency of recombinant hFIX, by enhancing its γ-carboxylation, which is inhibited by Calumenin (CALU), we used intronic artificial microRNAs (amiRNAs) for the CALU downregulation. METHODS: Two human CALU (hCALU)-specific amiRNAs were designed, validated and inserted within a truncated form of the hFIX intron 1, in either 3'- or 5'-untranslated regions of the hFIX cDNA, in an expression vector. After transfections of a human cell line with the recombinant constructs, processing of the miRNAs confirmed by RT-PCR, using stem-loop primers. The hFIX and hCALU expression assessments were done based on RT-PCR results. The Gamma(γ)-carboxylation of the expressed hFIX was examined by a barium citrate precipitation method, followed by Enzyme-Linked Immunosorbent Assay. RESULTS: Efficient CALU down regulations, with more than 30-fold decrease, occurred in the cells carrying either of the two examined the 3'-located amiRNAs. The CALU downregulation in the same cells doubled the FIX γ-carboxylation, although the transcription of the FIX decreased significantly. On the other hand, while the expression of the amiRNAs from the 5'-located intron had no decreasing effect on the expression level of CALU, the level of hFIX transcription in these cells increased almost twofold compared to the construct without amiRNA. CONCLUSION: The CALU downregulation, consistent with efficient hFIX γ-carboxylation, occurred in the cells carrying either of the two amiRNAs containing constructs, although it was affected by the locations of the amiRNA carrying introns, suggesting a possible need to optimize the conditions for the amiRNAs expression.

Aberration of the modulatory functions of intronic microRNA hsa-miR-933 on its host gene ATF2 results in type II diabetes mellitus and neurodegenerative disease development.

BACKGROUND: MicroRNAs are ~ 22-nucleotide-long biological modifiers that act as the post-transcriptional modulator of gene expression. Some of them are identified to be embedded within the introns of protein-coding genes, these miRNAs are called the intronic miRNAs. Previous findings state that these intronic miRNAs are co-expressed with their host genes. This co-expression is necessary to maintain the robustness of the biological system. Till to date, only a few experiments are performed discretely to elucidate the functional relationship between few co-expressed intronic miRNAs and their associated host genes. RESULTS: In this study, we have interpreted the underlying modulatory mechanisms of intronic miRNA hsa-miR-933 on its target host gene ATF2 and found that aberration can lead to several disease conditions. A protein-protein interaction network-based approach was adopted, and functional enrichment analysis was performed to elucidate the significantly over-represented biological functions and pathways of the common targets. Our approach delineated that hsa-miR-933 might control the hyperglycemic condition and hyperinsulinism by regulating ATF2 target genes MAP4K4, PRKCE, PEA15, BDNF, PRKACB, and GNAS which can otherwise lead to the development of type II diabetes mellitus. Moreover, we showed that hsa-miR-933 can regulate a target of ATF2, brain-derived neurotrophic factor (BDNF), to modulate the optimal expression of ATF2 in neuron cells to render neuroprotection for the inhibition of neurodegenerative diseases. CONCLUSIONS: Our in silico model provides interesting resources for experimentations in a model organism or cell line for further validation. These findings may extend the common perception of gene expression analysis with new regulatory functionality.

miR-378 and its host gene Ppargc1ß exhibit independent expression in mouse skeletal muscle.

MicroRNAs (miRNAs) are implicated in multiple biological processes in physiological and pathological settings. Nearly half of the known miRNAs are classified as 'intronic' miRNAs because they are embedded within the introns of protein-coding or noncoding genes. Such miRNAs were thought to be processed from primary host gene transcripts and share the promoter of their host. Recent analyses predicted that some intronic miRNAs might be transcribed and regulated as independent units, but there is little direct evidence for this in a specific biological context. Here, we focused on miR-378, which is located within the first intron of the peroxisome proliferator-activated receptor γ coactivator 1-beta (Ppargc1ß) gene and critically regulates skeletal muscle cell differentiation and muscle regeneration. We demonstrate that miR-378 and Ppargc1ß exhibit distinct expression patterns during skeletal muscle cell differentiation. In terminally differentiated adult skeletal muscle tissues of mice, miR-378 is predominantly expressed in glycolytic muscle, whereas Ppargc1ß is mainly expressed in oxidative soleus muscle. Mechanistically, miR-378, but not Ppargc1ß, is regulated by the transcription factor, MyoD, in muscle cells. Our findings identify a regulatory model of miR-378 expression, thereby helping us to understand its physiological function in skeletal muscle.

Polarity Acquisition in Cortical Neurons Is Driven by Synergistic Action of Sox9-Regulated Wwp1 and Wwp2 E3 Ubiquitin Ligases and Intronic miR-140.

The establishment of axon-dendrite polarity is fundamental for radial migration of neurons during cortex development of mammals. We demonstrate that the E3 ubiquitin ligases WW-Containing Proteins 1 and 2 (Wwp1 and Wwp2) are indispensable for proper polarization of developing neurons. We show that knockout of Wwp1 and Wwp2 results in defects in axon-dendrite polarity in pyramidal neurons, and their aberrant laminar cortical distribution. Knockout of miR-140, encoded in Wwp2 intron, engenders phenotypic changes analogous to those upon Wwp1 and Wwp2 deletion. Intriguingly, transcription of the Wwp1 and Wwp2/miR-140 loci in neurons is induced by the transcription factor Sox9. Finally, we provide evidence that miR-140 supervises the establishment of axon-dendrite polarity through repression of Fyn kinase mRNA. Our data delineate a novel regulatory pathway that involves Sox9-[Wwp1/Wwp2/miR-140]-Fyn required for axon specification, acquisition of pyramidal morphology, and proper laminar distribution of cortical neurons.

ANK1 Methylation regulates expression of MicroRNA-486-5p and discriminates lung tumors by histology and smoking status.

The intragenic tumor-suppressor microRNA miR-486-5p is often down-regulated in non-small cell lung cancer (NSCLC) but the mechanism is unclear. This study investigated epigenetic co-regulation of miR-486-5p and its host gene ANK1. MiR-486-5p expression in lung tumors and cell lines was significantly reduced compared to normal lung (p < 0.001) and is strongly correlated with ANK1 expression. In vitro, siRNA-mediated ANK1 knockdown in NSCLC cells also reduced miR-486-5p while the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced expression of both. ANK1 promoter CpG island was unmethylated in normal lung but methylated in 45% (118/262) lung tumors and 55% (17/31) NSCLC cell lines. After adjustment for tumor histology and smoking, methylation was significantly more prevalent in adenocarcinoma (101/200, 51%) compared to squamous cell carcinoma (17/62, 27%), p < 0.001; HR = 3.513 (CI: 1.818-6.788); and in smokers (73/128, 57%) than never-smokers (28/72, 39%), p = 0.014; HR = 2.086 (CI: 1.157-3.759). These results were independently validated using quantitative methylation data for 809 NSCLC cases from The Cancer Genome Atlas project. Together, our data indicate that aberrant ANK1 methylation is highly prevalent in lung cancer, discriminate tumors by histology and patients' smoking history, and contributes to miR-486-5p repression.

Discordance of MCM7 mRNA and its Intronic MicroRNA Levels Under Hypoxia.

BACKGROUND: Intronic microRNAs (miRNAs) are considered to be transcribed using their host gene promoter. However, about one third of intronic miRNAs are predicted to have independent promoter elements. MATERIALS AND METHODS: Human breast cancer cells were cultured under normoxia or hypoxia, and expression levels of intronic miR-106b-25 cluster miRNAs and their host gene minichromosome maintenance complex component 7 (MCM7) transcripts were analyzed by semi-quantitative polymerase chain reaction. The putative promoter element of miR-106b-25 cluster was analyzed by chromatin immunoprecipitation and luciferase assays. RESULTS: Exposure to hypoxia reduced the expression of MCM7 mRNA and a primary transcript of miR-106b-25 cluster, but did not affect that of mature miRNAs. The putative promoter element of miR-106b-25 cluster was not bound by hypoxia-inducible factor 1-alpha (HIF1-α), and was not activated under hypoxia. CONCLUSION: Maintenance of miR-106b-25 cluster miRNA levels under hypoxia was not caused by the activation of an independent promoter element.

The Supraspliceosome - A Multi-Task Machine for Regulated Pre-mRNA Processing in the Cell Nucleus.

Pre-mRNA splicing of Pol II transcripts is executed in the mammalian cell nucleus within a huge (21 MDa) and highly dynamic RNP machine - the supraspliceosome. It is composed of four splicing active native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA. Supraspliceosomes harbor protein splicing factors and all the five-spliceosomal U snRNPs. Recent analysis of specific supraspliceosomes at defined splicing stages revealed that they harbor all five spliceosomal U snRNAs at all splicing stages. Supraspliceosomes harbor additional pre-mRNA processing components, such as the 5'-end and 3'-end processing components, and the RNA editing enzymes ADAR1 and ADAR2. The structure of the native spliceosome, at a resolution of 20 Å, was determined by cryo-EM. A unique spatial arrangement of the spliceosomal U snRNPs within the native spliceosome emerged from in-silico studies, localizing the five U snRNPs mostly within its large subunit, and sheltering the active core components deep within the spliceosomal cavity. The supraspliceosome provides a platform for coordinating the numerous processing steps that the pre-mRNA undergoes: 5' and 3'-end processing activities, RNA editing, constitutive and alternative splicing, and processing of intronic microRNAs. It also harbors a quality control mechanism termed suppression of splicing (SOS) that, under normal growth conditions, suppresses splicing at abundant intronic latent 5' splice sites in a reading frame-dependent fashion. Notably, changes in these regulatory processing activities are associated with human disease and cancer. These findings emphasize the supraspliceosome as a multi-task master regulator of pre-mRNA processing in the cell nucleus.