Steroid Receptor RNA Activator (SRA) Modification by the Human Pseudouridine Synthase 1 (hPus1p): RNA Binding, Activity, and Atomic Model

Abstract

The most abundant of the modified nucleosides, and once considered as the “fifth” nucleotide in RNA, is pseudouridine, which results from the action of pseudouridine synthases. Recently, the mammalian pseudouridine synthase 1 (hPus1p) has been reported to modulate class I and class II nuclear receptor responses through its ability to modify the Steroid receptor RNA Activator (SRA). These findings highlight a new level of regulation in nuclear receptor (NR)-mediated transcriptional responses. We have characterised the RNA association and activity of the human Pus1p enzyme with its unusual SRA substrate. We validate that the minimal RNA fragment within SRA, named H7, is necessary for both the association and modification by hPus1p. Furthermore, we have determined the crystal structure of the catalytic domain of hPus1p at 2.0 Å resolution, alone and in a complex with several molecules present during crystallisation. This model shows an extended C-terminal helix specifically found in the eukaryotic protein, which may prevent the enzyme from forming a homodimer, both in the crystal lattice and in solution. Our biochemical and structural data help to understand the hPus1p active site architecture, and detail its particular requirements with regard to one of its nuclear substrates, the non-coding RNA SRA.

Towards structural classification of long non-coding RNAs

“…To demonstrate this principle, we consider the 873 nt steroid receptor RNA activator lncRNA in humans (SRA-1). This lncRNA co-activates the hormone response in human T-47D cells and co-immunoprecipitates with a large number of important proteins, including several hormone receptors (estrogen receptor, progesterone receptor, androgen receptor, glucocorticoid receptor and thyroid receptor) [67–70]. Binding assays in in vitro cell-free reconstituted systems have shown strong binding to the pseudouridinylase Pus1p, estrogen receptor, thyroid receptor, the sex reversal factor DAX-1, and the epigenetic factor SHARP.…”

New Insights Into the Long Non-coding RNA SRA: Physiological Functions and Mechanisms of Action

“…As described above, SRA regulates NR transcriptional activities through directly or indirectly interacting with NRs (Table 1 and Figure 3 ). SRA also interacts with other TFs, such as MyoD ( 62 ), Oct4 ( 61 ), Nanog ( 73 ), and FOXO1 ( 74 ); and forms a complex with coregulators of NRs and TFs, such as SRC-1 ( 23 ), P68 ( 62 , 69 ), PUS1/3 ( 71 , 75 ), components of RISC complex proteins (PACT, TRBP, Dicer, and Argonaut2) ( 72 ), and repressive coregulatory proteins SHARP ( 48 ) and SLIRP ( 47 ). This suggests that SRA works in concert with these interacting partners to regulate target gene expression.…”

Pseudouridine: Still mysterious, but never a fake (uridine)!

“…203 Positions with known or putative guide RNAs are depicted in blue, while gray positions await guide RNA identification. 203 Not shown are C-containing SRA RNA 204 and human telomerase RNA.…”