Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis

Detection of the HIV-1 Minus-Strand-Encoded Antisense Protein and Its Association with Autophagy

HIV-1 proteins are synthesized from a single transcript in an unspliced form or following splicing, but the existence of an antisense protein (ASP) expressed from an antisense polyadenylated transcript has been suggested. Difficulties linked to the detection of this protein in mammalian cells led us to codon optimize its cDNA. Codon-optimized ASP was indeed efficiently detected in various transfected cell lines following flow cytometry and confocal microscopy analyses. Western blot analyses also led to the detection of optimized ASP in transfected cells but also provided evidence of its instability and high multimerization potential. ASP was mainly distributed in the cytoplasm in a punctate manner, which was reminiscent of autophagosomes. In agreement with this observation, a significant increase in ASP-positive cells and loss of its punctate distribution was observed in transfected cells when autophagy was inhibited at early steps. Induction of autophagy was confirmed by Western blot analyses that showed an ASP-mediated increase in levels of LC3b-II and Beclin 1, as well as colocalization and interaction between ASP and LC3. Interestingly, Myc-tagged ASP was detected in the context of proviral DNA following autophagy inhibition with a concomitant increase in the level and punctate distribution of LC3b-II. Finally, 3-methyladenine treatment of transfected or infected U937 cells decreased extracellular p24 levels in wild-type proviral DNA and to a much lesser extent in ASP-mutated proviral DNA. This study provides the first detection of ASP in mammalian cells by Western blotting. ASP-induced autophagy might explain the inherent difficulty in detecting this viral protein and might justify its presumed low abundance in infected cells

An Endogenous RNA Transcript Antisense to CNGα1 Cation Channel mRNA

CNG channels are cyclic nucleotide-gated Ca(2+)-permeable channels that are suggested to be involved in the activity-dependent alterations of synaptic strength that are thought to underlie information storage in the CNS. In this study, we isolated an endogenous RNA transcript antisense to CNGα1 mRNA. This transcript was capable of down-regulating the expression of sense CNGα1 in the Xenopus oocyte expression system. RT-PCR, Northern blot, and in situ hybridization analyses showed that the transcript was coexpressed with CNGα1 mRNA in many regions of human brain, notably in those regions that were involved in long-term potentiation and long-term depression, such as hippocampal CA1 and CA3, dentate gyrus, and cerebellar Purkinje layer. Comparison of expression patterns between adult and fetal cerebral cortex revealed that there were concurrent developmental changes in the expression levels of anti-CNG1 and CNGα1. Treatment of human glioma cell T98 with thyroid hormone T(3) caused a significant increase in anti-CNG1 expression and a parallel decrease in sense CNGα1 expression. These data suggest that the suppression of CNGα1 expression by anti-CNG1 may play an important role in neuronal functions, especially in synaptic plasticity and cortical development. Endogenous antisense RNA-mediated regulation may represent a new mechanism through which the activity of ion channels can be regulated in the human CNS