RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3′-Tail of the Long Non-coding RNA BC200 (BCYRN1)

Abstract

RNA helicase associated with AU-rich element (RHAU) is an ATP-dependent RNA helicase that demonstrates high affinity for quadruplex structures in DNA and RNA. To elucidate the significance of these quadruplex-RHAU interactions, we have performed RNA co-immunoprecipitation screens to identify novel RNAs bound to RHAU and characterize their function. In the course of this study, we have identified the non-coding RNA BC200 (BCYRN1) as specifically enriched upon RHAU immunoprecipitation. Although BC200 does not adopt a quadruplex structure and does not bind the quadruplex-interacting motif of RHAU, it has direct affinity for RHAU in vitro. Specifically designed BC200 truncations and RNase footprinting assays demonstrate that RHAU binds to an adenosine-rich region near the 3-end of the RNA. RHAU truncations support binding that is dependent upon a region within the C terminus and is specific to RHAU isoform 1. Tests performed to assess whether BC200 interferes with RHAU helicase activity have demonstrated the ability of BC200 to act as an acceptor of unwound quadruplexes via a cytosine-rich region near the 3-end of the RNA. Furthermore, an interaction between BC200 and the quadruplex-containing telomerase RNA was confirmed by pull-down assays of the endogenous RNAs. This leads to the possibility that RHAU may direct BC200 to bind and exert regulatory functions at quadruplex-containing RNA or DNA sequences.RNA helicase associated with AU-rich element (RHAU), 8 also known as DHX36 and G4R1, is an ATP-dependent DEAHbox RNA helicase initially characterized as a regulator of mRNA stability via binding to the AU-rich element of urokinase plasminogen activator mRNA (1). Subsequent to this, the discovery that RHAU possesses the ability to unwind both DNA and RNA quadruplexes shifted the focus of RHAU to a role in quadruplex biology (2-4).Quadruplexes are stable secondary structures that occur in guanine-rich nucleic acids through the alignment and hydrogen bonding of guanines in multiple tetrad planes (5). A typical requirement for quadruplex formation is the presence of four tracts of three or more guanines that align in parallel or antiparallel strands with short (Ͻ7-nt) interspersed loops (6); however, quadruplexes with longer loops have also been reported (7,8). Quadruplexes have been shown to possess myriad roles in gene transcription and post-transcriptional regulation of both coding and non-coding RNAs (9 -16).The quadruplex binding specificity of RHAU is mediated by a short N-terminal motif, referred to as the RHAU-specific motif (RSM). In addition to the RSM, RHAU contains a catalytic helicase core (DEXDc and HELICc domains) as well as an HA2 domain and oligosaccharide binding (OB) fold. Whereas the RSM confers quadruplex interacting specificity, maximal quadruplex binding affinity requires the presence of the helicase core (17, 18). The DHX36 gene generates two known splice variants of RHAU. Whereas isoform 1 localizes to both nucleus and cytoplasm, isoform 2 lacks 14 amino acids within the helicase domain and is p...

Regulation of BC200 RNA‐mediated translation inhibition by hnRNP E1 and E2

“…The cleavage patterns obtained in the absence of hnRNP E proteins agreed well with the predicted secondary structure [38], except that nucleotides 130-132, 142, 143, 165, 166, and 176-181 in the A-rich and Crich domains were RNase V1 sensitive (Figs 5 and 6). The cleavage patterns obtained in the absence of hnRNP E proteins agreed well with the predicted secondary structure [38], except that nucleotides 130-132, 142, 143, 165, 166, and 176-181 in the A-rich and Crich domains were RNase V1 sensitive (Figs 5 and 6).…”

“…To explore a possible mechanistic basis for the differential binding of hnRNP E1 and E2 to BC200 RNA, we performed an RNA footprinting analysis using RNase V1, RNase A, RNase T1, and the Pb(II) ion. The cleavage patterns obtained in the absence of hnRNP E proteins agreed well with the predicted secondary structure [38], except that nucleotides 130-132, 142, 143, 165, 166, and 176-181 in the A-rich and Crich domains were RNase V1 sensitive (Figs 5 and 6). Since RNase V1 cleaves both double-stranded and stacked single-stranded RNA regions, the RNase V1protected regions in the A-rich and C-rich domains are likely to contain highly stacked structures.…”

“…BC200 RNA can be dissected into three domains: a 5 0 Alu domain, a central A-rich domain, and a unique 3 0 C-rich domain [38]. BC200 RNA can be dissected into three domains: a 5 0 Alu domain, a central A-rich domain, and a unique 3 0 C-rich domain [38].…”

“…To delineate the binding domain(s) through which BC200 RNA interacts with hnRNP E1 and E2, we generated truncated domain derivatives of BC200 RNA. BC200 RNA can be dissected into three domains: a 5 0 Alu domain, a central A-rich domain, and a unique 3 0 C-rich domain [38]. We thus generated four domain derivatives: the Alu domain (nts: 1-121), the A/C-rich domain (nts: 122-200), the A-rich domain (nts: 122-161), and the C-rich domain (nts: 162-200).…”

Long Noncoding RNA BCYRN1 Promotes the Proliferation of Colorectal Cancer Cells via Up-Regulating NPR3 Expression

“…Dysregulation of lncRNA BCYRN1 has been found in various cancers, as well as in neurodegenerative diseases [18,19]. However, the clinical role of BCYRN1 and its biological functions in CRC remains unknown.…”