Deficiency in the Ubiquitin Conjugating Enzyme UBE2A in Alzheimer’s Disease (AD) is Linked to Deficits in a Natural Circular miRNA-7 Sponge (circRNA; ciRS-7)

G C A T T A C G G C A T genes Communication Deficiency in the Ubiquitin Conjugating Enzyme UBE2A in Alzheimer’s Disease (AD) is Linked to Deficits in a Natural Circular miRNA-7 Sponge (circRNA; ciRS-7) Yuhai Zhao 1,2 , Peter N. Alexandrov 3 , Vivian Jaber 1 and Walter J. Lukiw 1,4,5, * 1 2 3 4 5 * LSU Neuroscience Center, Louisiana State University Health Science Center, New Orleans, LA 70112, USA; (Y.Z.); (V.J.) Department of Anatomy and Cell Biology, Louisiana State University Health Science Center, New Orleans, LA 70112, USA Russian Academy of Medical Sciences, Moscow 113152, Russia; Department of Ophthalmology, Louisiana State University Health Science Center, New Orleans, LA 70112, USA Department of Neurology, Louisiana State University Health Science Center, New Orleans, LA 70112, USA Correspondence: ; Tel.: +1-504-599-0842 Academic Editor: George A. Calin Received: 17 October 2016; Accepted: 30 November 2016; Published: 5 December 2016 Abstract: Our understanding of the highly specialized functions for small non-coding single-stranded RNA (ssRNA) in the transcriptome of the human central nervous system (CNS) continues to evolve. Circular RNAs (circRNAs), a recently discovered class of ssRNA enriched in the brain and retina, are extremely stable and intrinsically resilient to degradation by exonuclease. Conventional methods of ssRNA, microRNA (miRNA), or messenger RNA (mRNA) detection and quantitation requiring free ribonucleotide ends may have considerably underestimated the quantity and significance of CNS circRNA in the CNS. Highly-specific small ssRNAs, such as the ~23 nucleotide (nt) Homo sapien microRNA-7 (hsa-miRNA-7; chr 9q21.32), are not only abundant in the human limbic system but are, in addition, associated with a ~1400 nt circRNA for miRNA-7 (ciRS-7) in the same anatomical region. Structurally, ciRS-7 contains about ~70 tandem anti-miRNA-7 sequences and acts as an endogenous, anti-complementary miRNA-7 “sponge” that attracts, binds, and, hence, quenches, natural miRNA-7 functions. Using a combination of DNA and miRNA array technologies, enhanced LED-Northern and Western blot hybridization, and the magnesium-dependent exoribonuclease and circRNA-sensitive probe RNaseR, here we provide evidence of a significantly misregulated ciRS-7-miRNA-7-UBE2A circuit in sporadic Alzheimer’s disease (AD) neocortex (Brodmann A22) and hippocampal CA1. Deficits in ciRS-7-mediated “sponging events”, resulting in excess ambient miRNA-7 appear to drive the selective down-regulation in the expression of miRNA-7-sensitive mRNA targets, such as that encoding the ubiquitin conjugating enzyme E2A (UBE2A; chr Xq24). UBE2A, which normally serves as a central effector in the ubiquitin-26S proteasome system, coordinates the clearance of amyloid peptides via proteolysis, is known to be depleted in sporadic AD brain and, hence, contributes to amyloid accumulation and the formation of senile plaque deposits. Dysfunction of circRNA-miRNA-mRNA regulatory systems appears to represent another important layer of epigenetic control over pathogenic gene expression programs in the human CNS that are targeted by the sporadic AD process. Keywords: Alzheimer’s disease (AD); circular RNA (circRNA); genetic control; miRNA-7; proteasome; proteolysis; ubiquitin conjugating enzyme E2A (UBE2A) Genes 2016, 7, 116; doi:10.3390/genes7120116 www.mdpi.com/journal/genes Genes 2016, 7, 116 2 of 9 1. Introduction In eukaryotic cells the post-translational modification of end-stage proteins with ubiquitin is an important cellular regulatory mechanism for the targeting and shuttling of abnormal, short-lived proteins, transcription factors, and/or neurotoxic proteins destined for degradation and proteolysis into the ubiquitin-26S proteasome system [1–3]. As such, ubiquitin-mediated tagging, trafficking, and elimination of “waste” proteins plays a crucial role in cell-cycle regulation, DNA repair, cell growth, vesicular transport and immune function, and dysfunction of the ubiquitin-26S proteasome pathway is known to contribute to cancer, and immunological and neurodegenerative disorders [2–7]. The ubiquitination signaling pathway involves at least three categories of enzymes: a ubiquitin-activating enzyme UBE1, a ubiquitin-conjugating enzyme UBE2A, and a ubiquitin-protein ligase UBE3 [3–6]. The ubiquitin conjugating enzyme E2A (UBE2A) is part of an UBE2 enzyme group that catalyzes the transfer of ubiquitin from UBE1 to the active site cysteine of the UBE2A via a trans-thioesterification reaction and occupies a central regulatory position in the ubiquitination mechanism [3–6]. Interestingly, UBE2A (encoded at chr Xq24) is associated with neurological diseases that involve cognitive disruption, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), mental retardation, X-linked syndrome, X-linked intellectual disability (Nascimento type), and other progressive, age-related neurodegenerative disorders [2–6]. Current studies involving bioinformatics analysis of microRNA-messenger RNA (miRNA-mRNA) coupling in the aging human brain indicate a significant miRNA-7-UBE2A-mRNA-30 -UTR interaction [7–13]. These studies, using multiple methods of analysis, subsequently uncovered a novel circular RNA (circRNA) for the miRNA-7 (ciRS-7) mechanism coupled to an ambient increase in miRNA-7 in the sporadic AD hippocampal CA1 region and superior temporal lobe neocortex (Brodmann A22) [7–13]; this may in part explain: (i) an expanding pathological role for increased miRNA-7 in inflammatory degeneration [14,15]; (ii) the down-regulation of UBE2A protein, a central effector in the ubiquitin-26S proteasome proteolysis and clearance system [3–6]; and (iii) the consequences of deficits in UBE2A and the ubiquitin-26S proteasome proteolysis, which are the accumulation and aggregation of brain waste products, such as amyloid proteins that are characteristic of the pathogenic lesions that progressively accumulate in the AD brain [16–18]. 2. Materials and Methods 2.1. Reagents, Control, and Alzheimer’s Disease (AD) Brain Tissues Except as indicated, reagents used in these experiments were obtained from independent commercial suppliers and were used without further purification. RNaseR, a magnesium-dependent hydrolytic 30 →50 exoribonuclease that digests essentially all linear RNAs but not lariat or circular RNA structures, or double-stranded RNA with 30 -overhangs shorter than seven nucleotides was purchased from Epicentre (RNR07250 Illumina, Madison, WI, USA) and used according to established RnaseR protocols [5,10,19]. Alzheimer’s disease (AD) and age-matched control human temporal lobe and hippocampal CA1 were obtained from brain and tissue repositories, including the Institute for Memory Impairments and Neurological Disorders and the University of California at Irvine (UCI and our own archives); tissues were analyzed for total miRNA using miRNA arrays and LED-Northern blots and CFH abundance using Western blot analy (...truncated)