Distinct RNA motifs are important for coactivation of steroid hormone receptors by steroid receptor RNA activator (SRA)

Abstract

Steroid receptor RNA activator (SRA) is an RNA transcript that functions as a eukaryotic transcriptional coactivator for steroid hormone receptors. We report here the isolation and functional characterization of distinct RNA substructures within the SRA molecule that constitute its coactivation function. We used comparative sequence analysis and free energy calculations to systematically study SRA RNA subdomains for identification of structured regions and base pairings, and we used site-directed mutagenesis to assess their functional consequences. Together with genetic deletion analysis, this approach identified six RNA motifs in SRA important for coactivation. Because all nucleotide changes in the mutants that disrupted SRA function were silent mutations presumed not to alter deduced encoded amino acid sequence, our analysis provides strong evidence that SRA-mediated coactivation is executed by distinct RNA motifs and not by an encoded protein.N uclear receptors play critical roles in eukaryotic development, metabolism, reproduction, and disease through regulation of gene expression (1, 2). Recent advances in transcription have suggested that nuclear receptors form large, multicomponent complexes with coactivators and corepressors at the enhancer and promoter regions of target genes (3, 4). These complexes include factors exerting different enzymatic functions such as chromatin remodeling activities, and a variety of combinations of coregulators are believed to be essential for regulated gene expression.We have previously reported the isolation and functional characterization of a novel transcriptional coactivator termed SRA (steroid receptor RNA activator; ref. 5). SRA acts as a catalytic RNA transcript by regulating eukaryotic gene expression mediated by the steroid receptors (SRs). When overexpressed in mammalian cells, recombinant SRA showed potent coactivation activity with the receptors for androgens, estrogens, glucocorticoids, and progestins (PR). We showed that several isoforms of SRA exist, and we grouped them into three splice classes based on their sequences outside a common core region. The SRA sequences of all of our cDNA clones contain an ORF but are devoid of an initiation ATG. Although the existence of a translation product of SRA cannot be categorically excluded, we have provided evidence to indicate that SRA functions as an RNA transcript (5).Because the function of an RNA can be best understood in terms of its secondary or tertiary structure, we wished to further define the coactivation function we previously observed in SRA by using low-resolution RNA structure modeling. RNA secondary structure is a composite of hydrogen bonds between bases allowing certain noncanonical pairings, forming structures with double-helical motifs, bulges, bubbles, and loops that then spatially arrange to assemble specific intra-and intermolecular interactions. By using comparative sequence analysis and lowresolution RNA modeling, we set out to predict functional substructures of SRA and to obtain a framework wi...

The steroid receptor RNA activator protein is expressed in breast tumor tissues

“…To our knowledge, all the SRA sequences used by others when investigating SRA RNA function lacked the first 2 starting methionines and were consequently unable to encode SRAP. [5][6][7]11 The SRA sequence used here contains 32 additional 5 0 end base pairs with 2 putative starting methionines and therefore has the capacity to initiate the translation of either a 236-or a 224-amino acid SRAP. 14 This coding RNA is expected to function as an ER-activating RNA since it contains an intact SRA core sequence previously shown to be necessary and suffi- cient for SRA RNA to function as a non-coding RNA.…”

“…5 SRA mechanisms of action have since become the focus of extensive investigation. SRA was shown to contain a core RNA sequence necessary and sufficient to mediate steroid receptor activity 6 through interactions with several proteins including the coactivator/corepressor SHARP, 7 SRC1, 5 and the AF-1-specific activator p72/p68 protein. 8 Post-transcriptional modifications of SRA have also been shown to participate in the ability of this RNA to modulate receptor activity.…”

The four Rs of RNA-directed evolution

“…Many pathways involve trans-acting coRNAs that affect transcriptional initiation and elongation. Examples include the steroid receptor RNA activator 58 , dsRNA motifs bound by the transcriptional regulator NF110 (ref. 59), the U1 small nuclear RNA that associates with TFIIH 60 and the 7SK RNA that inhibits Cdk9 (the RNA polymerase II C-terminal domain kinase) 61,62 .…”

The steroid receptor RNA activator protein (SRAP) controls cancer cell migration/motility

“…secondary structure of SRA, necessary for its RNA action, but do not interfere with its coding properties [9,22,28]. In order to easily identify, sort and isolate cells overexpressing SRAP, this ''only-SRAP" construct was cloned in frame with GFP.…”