SRA的RNA和蛋白质均有转录辅激活功能

类固醇受体激活物（steroid receptor activator, SRA）是一种 类固醇受体辅激活物.最初的研究认为,SRA只存在RNA形式,不存在蛋白质 形式.但是后来的研究发现,SRA是在RNA和蛋白质两个水平上发挥功能的分 子,其cDNA序列存在687 bp保守的核心区域,该核心区域对其发挥转录共激 活活性是必需的.SRA的RNA形式主要参与核受体的转录共激活作用,其表达 与乳腺癌的发生有很大关系,SRA的蛋白质形式（steroid receptor activator protein ,SRAP）也具有类似的功能.但是不同于RNA形式, SRAP可结合到特定基因的启动子区域,并起到阻遏物的作用.本文对SRA的 特点、表达及功能等方面的最新研究进展及其可能的作用机制与作用形式 进行概述.     

Human DND1‐RRM2 forms a non‐canonical domain swapped dimer

RNA recognition motif (RRM) being the most abundant RNA binding domain in eukaryotes, is a major player in cellular regulation. Several variations in the canonical βαββαβ topology have been observed. We have determined the 2.3 Å crystal structure of the human DND1‐RRM2 domain. The structure revealed an interesting non‐canonical RRM fold, which is maintained by the formation of a 3D domain swapped dimer between β₁ and β₄ strands across protomers. We have delineated the structural basis of the stable domain swapped dimer formation using the residue level dynamics of protein explored by NMR spectroscopy and MD simulations. Our structural and dynamics studies substantiate major determinants and molecular basis for domain swapped dimerization observed in the RRM domain.     

SLIRP, a small SRA binding protein, is a nuclear receptor corepressor

Steroid receptor RNA activator (SRA), the only known RNA coactivator, augments transactivation by nuclear receptors (NRs). We identified SLIRP (SRA stem-loop interacting RNA binding protein) binding to a functional substructure of SRA, STR7. SLIRP is expressed in normal and tumor tissues, contains an RNA recognition motif (RRM), represses NR transactivation in a SRA- and RRM-dependent manner, augments the effect of Tamoxifen, and modulates association of SRC-1 with SRA. SHARP, a RRM-containing corepressor, also binds STR7, augmenting repression with SLIRP. SLIRP colocalizes with SKIP (Chr14q24.3), another NR coregulator, and reduces SKIP-potentiated NR signaling. SLIRP is recruited to endogenous promoters (pS2 and metallothionein), the latter in a SRA-dependent manner, while NCoR promoter recruitment is dependent on SLIRP. The majority of the endogenous SLIRP resides in the mitochondria. Our data demonstrate that SLIRP modulates NR transactivation, suggest it may regulate mitochondrial function, and provide mechanistic insight into interactions between SRA, SLIRP, SRC-1, and NCoR.     

Site-directed spin labeling electron paramagnetic resonance study of the ORF1 protein from a mouse L1 retrotransposon

Long interspersed nuclear element-1 is a highly abundant mammalian retrotransposon that comprises 17% of the human genome. L1 retrotransposition requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. ORF1p has been shown to adopt a homo-trimeric, asymmetric dumbbell-shaped structure. However, its atomic-level structure and mechanism of RNA binding remains poorly understood. Here, we report the results of a site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) study of 27 residues within the RNA binding region of the full-length protein. The EPR data are compatible with the large RNA binding lobe of ORF1p containing a RNA recognition motif (RRM) domain and a carboxyl-terminal domain (CTD) that are predicted from crystallographic and NMR studies of smaller fragments of the protein. Interestingly, the EPR data indicate that residues in strands β3 and β4 of the RRM are structurally unstable, compatible with the previously observed sensitivity of this region to proteolysis. Affinity measurements and RNA-dependent EPR spectral changes map the RNA binding site on ORF1p to residues located in strands β3 and β4 of the RRM domain and to helix α1 of the CTD. Complementary in vivo studies also identify residues within the RRM domain that are required for retrotransposition. We propose that in the context of the full-length trimeric protein these distinct surfaces are positioned adjacent to one another providing a continuous surface that may interact with nucleic acids.     

In Human Pseudouridine Synthase 1 (hPus1), a C-Terminal Helical Insert Blocks tRNA from Binding in the Same Orientation as in the Pus1 Bacterial Homologue TruA,Consistent with Their Different Target Selectivities

Human pseudouridine (Ψ) synthase Pus1 (hPus1) modifies specific uridine residues in several non-coding RNAs: tRNA, U2 spliceosomal RNA, and steroid receptor activator RNA. We report three structures of the catalytic core domain of hPus1 from two crystal forms, at 1.8 Å resolution. The structures are the first of a mammalian Ψ synthase from the set of five Ψ synthase families common to all kingdoms of life. hPus1 adopts a fold similar to bacterial Ψ synthases, with a central antiparallel β-sheet flanked by helices and loops. A flexible hinge at the base of the sheet allows the enzyme to open and close around an electropositive active-site cleft. In one crystal form, a molecule of Mes [2-(N-morpholino)ethane sulfonic acid] mimics the target uridine of an RNA substrate. A positively charged electrostatic surface extends from the active site towards the N-terminus of the catalytic domain, suggesting an extensive binding site specific for target RNAs. Two α-helices C-terminal to the core domain, but unique to hPus1, extend along the back and top of the central β-sheet and form the walls of the RNA binding surface. Docking of tRNA to hPus1 in a productive orientation requires only minor conformational changes to enzyme and tRNA. The docked tRNA is bound by the electropositive surface of the protein employing a completely different binding mode than that seen for the tRNA complex of the Escherichia coli homologue TruA.     

Structural characterization of Set1 RNA recognition motifs and their role in histone H3 lysine 4 methylation

The yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains, in addition to its catalytic SET domain, a conserved RNA recognition motif (RRM1). We present here the crystal structure and the secondary structure assignment in solution of the Set1 RRM1. Although RRM1 has the expected betaalphabetabetaalphabeta RRM-fold, it lacks the typical RNA-binding features of these modules. RRM1 is not able to bind RNA by itself in vitro, but a construct combining RRM1 with a newly identified downstream RRM2 specifically binds RNA. In vivo, H3K4 methylation is not affected by a point mutation in RRM2 that preserves Set1 stability but affects RNA binding in vitro. In contrast mutating RRM1 destabilizes Set1 and leads to an increase of dimethylation of H3K4 at the 5'-coding region of active genes at the expense of trimethylation, whereas both, dimethylation decreases at the 3'-coding region. Taken together, our results suggest that Set1 RRMs bind RNA, but Set1 RNA-binding activity is not linked to H3K4 methylation.     

Structure of the central RNA recognition motif of human TIA-1 at 1.95A resolution

T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95 Å resolution. Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1-RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein-protein interactions mediated by the RNP motifs of TIA-1-RRM2. Importantly, the TIA-1-RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding.     

Improved segmental isotope labeling methods for the NMR study of multidomain or large proteins: application to the RRMs of Npl3p and hnRNP L

The study of multidomain or large proteins in solution by NMR spectroscopy has been made possible in recent years by the development of new spectroscopic methods. However, resonance overlap found in large proteins remains a limiting factor, making resonance assignments and structure determination of large proteins very difficult. In this study, we present an expressed protein ligation protocol that can be used for the segmental isotopic labeling of virtually any multidomain or high molecular mass protein, independent of both the folding state and the solubility of the protein fragments, as well as independent of whether the fragments are interacting. The protocol was applied successfully to two different multidomain proteins containing RNA recognition motifs (RRMs), heterogeneous nuclear ribonucleoprotein L and Npl3p. High yields of segmentally labeled proteins could be obtained, allowing characterization of the interdomain interactions with NMR spectroscopy. We found that the RRMs of heterogeneous nuclear ribonucleoprotein L interact, whereas those of Npl3p are independent. Subsequently, the structures of the two RRMs of Npl3p were determined on the basis of samples in which each RRM was expressed individually. The two Npl3p RRMs adopt the expected βαββαβ fold.     

LncRNA SRA mediates cell migration,invasion, and progression of ovarian cancer via NOTCH signaling and epithelial–mesenchymal transition

Long non-coding RNA (lncRNA) is a newly identified regulator of tumor formation and tumor progression. The function and expression of lncRNAs remain to be fully elucidated, but recent studies have begun to address their importance in human health and disease. The lncRNA, SRA, known as steroid receptor activator, acts as an important modulator of gynecological cancer, and its expression may affect biological functions including proliferation, apoptosis, steroid formation, and muscle development. However, it is still not well known whether SRA is involved in the regulation of ovarian cancer. The present study investigated the molecular function and association between SRA expression and clinicopathological factors. In ovarian cancer cell lines, SRA knockdown and overexpression regulated cell migration, proliferation, and invasion. Both in vivo and in vitro experiments using knockdown and overexpression showed that SRA potently regulated epithelial–mesenchymal transition (EMT) and NOTCH pathway components.Further, clinical data confirmed that SRA was a significant predictor of overall survival (OS) and progression-free survival and patients with ovarian cancer exhibiting high expression of SRA exhibited higher recurrence rates than patients with low SRA expression. In conclusion, the present study indicates that SRA has clinical significance as its expression can predict the prognosis of ovarian cancer patients. High expression of the lncRNA SRA is strongly correlated with recurrence-free survival of ovarian cancer patients.