Long non-coding RNA (lncRNA) genes encode non-messenger RNAs that lack open reading frames (ORFs) longer than 300 nucleotides, lack evolutionary conservation in their shorter ORFs, and do not belong to any classical non-coding RNA category. LncRNA genes equal, or exceed in number, protein-coding genes in mammalian genomes. Most mammalian genomes harbor ~20,000 protein-coding genes that give rise to conventional messenger RNA (mRNA) transcripts. These coding genes exhibit sweeping evolutionary conservation in their ORFs. LncRNAs function via different mechanisms, including but not limited to: (1) serving as “enhancer” RNAs regulating nearby coding genes in cis; (2) functioning as scaffolds to create ribonucleoprotein (RNP) complexes; (3) serving as sponges for microRNAs; (4) acting as ribo-mimics of consensus transcription factor binding sites in genomic DNA; (5) hybridizing to other nucleic acids (mRNAs and genomic DNA); and, rarely, (6) as templates encoding small open reading frames (smORFs) that may encode short proteins. Any given lncRNA may have more than one of these functions. This review focuses on one fascinating case—the growth-arrest-specific (GAS)-5 gene, encoding a complicated repertoire of alternatively-spliced lncRNA isoforms. GAS5 is also a host gene of numerous small nucleolar (sno) RNAs, which are processed from its introns. Publications about this lncRNA date back over three decades, covering its role in cell proliferation, cell differentiation, and cancer. The GAS5 story has drawn in contributions from prominent molecular geneticists who attempted to define its tumor suppressor function in mechanistic terms. The evidence suggests that rodent Gas5 and human GAS5 functions may be different, despite the conserved multi-exonic architecture featuring intronic snoRNAs, and positional conservation on syntenic chromosomal regions indicating that the rodent Gas5 gene is the true ortholog of the GAS5 gene in man and other apes. There is no single answer to the molecular mechanism of GAS5 action. Our goal here is to summarize competing, not mutually exclusive, mechanistic explanations of GAS5 function that have compelling experimental support.

Aims: Causal transcripts at genomic loci associated with type 2 diabetes (T2D) are mostly unknown. The chr8p23.1 variant rs4841132, associated with an insulin-resistant diabetes risk phenotype, lies in the second exon of a long non-coding RNA (lncRNA) gene, LOC157273, located 175 kilobases from PPP1R3B, which encodes a key protein regulating insulin-mediated hepatic glycogen storage in humans. We hypothesized that LOC157273 regulates expression of PPP1R3B in human hepatocytes. Methods: We tested our hypothesis using Stellaris fluorescent in situ hybridization to assess subcellular localization of LOC157273; small interfering RNA (siRNA) knockdown of LOC157273, followed by RT-PCR to quantify LOC157273 and PPP1R3B expression; RNA-seq to quantify the whole-transcriptome gene expression response to LOC157273 knockdown; and an insulin-stimulated assay to measure hepatocyte glycogen deposition before and after knockdown. Results: We found that siRNA knockdown decreased LOC157273 transcript levels by approximately 80%, increased PPP1R3B mRNA levels by 1.7-fold, and increased glycogen deposition by >50% in primary human hepatocytes. An A/G heterozygous carrier (vs. three G/G carriers) had reduced LOC157273 abundance

Long non-coding RNAs (lncRNAs) are transcripts of a recently discovered class of genes which do not code for proteins. LncRNA genes are approximately as numerous as protein-coding genes in the human genome. However, comparatively little remains known about lncRNA functions. We globally interrogated changes in the lncRNA transcriptome of oestrogen receptor positive human breast cancer cells following treatment with oestrogen, and identified 127 oestrogen-responsive lncRNAs. Consistent with the emerging evidence that most human lncRNA genes lack homologues outside of primates, our evolutionary analysis revealed primate-specific lncRNAs downstream of oestrogen signalling. We demonstrate, using multiple functional assays to probe gain- and loss-of-function phenotypes in two oestrogen receptor positive human breast cancer cell lines, that two primate-specific oestrogen-responsive lncRNAs identified in this study (the oestrogen-repressed lncRNA BC041455, which reduces cell viability, and the oestrogen-induced lncRNA CR593775, which increases cell viability) exert previously unrecognized functions in cell proliferation and growth factor signalling pathways. The results suggest that oestrogen-responsive lncRNAs are capable of altering the proliferation and viability of human breast cancer cells. No effects on cellular phenotypes were associated with control transfections. As heretofore unappreciated components of key signalling pathways in cancers, including the MAP kinase pathway, lncRNAs hence represent a novel mechanism of action for oestrogen effects on cellular proliferation and viability phenotypes. This finding warrants further investigation in basic and translational studies of breast and potentially other types of cancers, has broad relevance to lncRNAs in other nuclear hormone receptor pathways, and should facilitate exploiting and targeting these cell viability modulating lncRNAs in post-genomic therapeutics.

Iron homeostasis is critical for cellular and organismal function and is tightly regulated to prevent toxicity or anemia due to iron excess or deficiency, respectively. However, subcellular regulatory mechanisms of iron remain largely unexplored. Here, we report that SEL1L-HRD1 protein complex of endoplasmic reticulum (ER)-associated degradation (ERAD) in hepatocytes controls systemic iron homeostasis in a ceruloplasmin (CP)-dependent, and ER stress-independent, manner. Mice with hepatocyte-specific Sel1L deficiency exhibit altered basal iron homeostasis and are sensitized to iron deficiency while resistant to iron overload. Proteomics screening for a factor linking ERAD deficiency to altered iron homeostasis identifies CP, a key ferroxidase involved in systemic iron distribution by catalyzing iron oxidation and efflux from tissues. Indeed, CP is highly unstable and a bona fide substrate of SEL1L-HRD1 ERAD. In the absence of ERAD, CP protein accumulates in the ER and is shunted to refolding, leading to elevated secretion. Providing clinical relevance of these findings, SEL1L-HRD1 ERAD is responsible for the degradation of a subset of disease-causing CP mutants, thereby attenuating their pathogenicity. Together, this study uncovers the role of SEL1L-HRD1 ERAD in systemic iron homeostasis and provides insights into protein misfolding-associated proteotoxicity.

8Aims: Causal transcripts at genomic loci associated with type 2 diabetes are mostly unknown. The 9chr8p23.1 variant rs4841132, associated with an insulin resistant diabetes risk phenotype, lies in the 10 second exon of a long non-coding RNA (lncRNA) gene, LOC157273, located 175 kilobases from 11 PPP1R3B, which encodes a key protein regulating insulin-mediated hepatic glycogen storage in 12humans. We hypothesized that LOC157273 regulates expression of PPP1R3B in human hepatocytes. 13 14Methods: We tested our hypothesis using Stellaris fluorescent in-situ hybridization to assess subcellular 15 localization of LOC157273; siRNA knockdown of LOC157273, followed by RT-PCR to quantify 16 LOC157273 and PPP1R3B expression; RNA-seq to quantify the whole-transcriptome gene expression 17 response to LOC157273 knockdown and an insulin-stimulated assay to measure hepatocyte glycogen 18 deposition before and after knockdown. 19 20Results: We found that siRNA knockdown decreased LOC157273 transcript levels by approximately 21 80%, increased PPP1R3B mRNA levels by 1.7-fold and increased glycogen deposition by >50% in 22 primary human hepatocytes. An A/G heterozygous carrier (vs. three G/G carriers) had reduced 23LOC157273 abundance due to reduced transcription of the A allele and increased PPP1R3B expression 24 and glycogen deposition. 25 26Conclusion: We show that the lncRNA LOC157273 is a negative regulator of PPP1R3B expression and 27 glycogen deposition in human hepatocytes and the causal transcript at an insulin resistant type 2 diabetes 28 risk locus. 29 30 31 32 33 42 (NR_040039.1:n.548A>G; reference allele A has frequency ~11%) was significantly associated with an 43 insulin resistance phenotype characterized by increased levels of fasting glucose (FG) and insulin (FI), 44 elevated levels of triglycerides and an increased waist-hip ratio (4). This chromosome 8 locus is highly 45 pleiotropic; and rs4841132 and nearby SNPs have been consistently associated with increased T2D risk 46as well as T2D-related metabolic phenotypes including glycemia in pregnancy, obesity, HDL:LDL ratio, 47total cholesterol, triglycerides, c-reactive protein levels, coronary artery disease, subclinical 48 atherosclerosis, and fatty liver disease (4,(9)(10)(11)(12)(13)(14)(15). 49 50The variant rs4841132 resides ~175 kb from the nearest protein-coding gene, PPP1R3B. PPP1R3B 51 encodes the glycogen-targeting subunit of PP1 protein phosphatase and is expressed most strongly in 52 liver in both rodents and man; and at lower levels in skeletal muscle and other tissues (16-18). PPP1R3B 53 connects ambient insulin to hepatic glycogen regulation: its overexpression in hepatocytes markedly 54 increases both basal and insulin-stimulated glycogen synthesis (19). PPP1R3B has long been an 55 attractive target for diabetes therapy, based on the concept of tipping ambient glycemic balance towards 56 hepatic glycogen deposition (17, 20). 57 58We previously localized rs4841132 to exon 2 of a previously unannotated long non-coding RNA 59 (lncRNA) gene, LOC157273 (...