Interaction of the Histone mRNA Hairpin with Stem–Loop Binding Protein (SLBP) and Regulation of the SLBP–RNA Complex by Phosphorylation and Proline Isomerization

In metazoans, the majority of histone proteins are generated from replication-dependent histone mRNAs. These mRNAs are unique in that they are not polyadenylated but have a stem-loop structure in their 3′ untranslated region. An early event in 3′ end formation of histone mRNAs is the binding of Stem-Loop Binding Protein (SLBP) to the stem-loop. Here we provide insight into the mechanism by which SLBP contacts the histone mRNA. There are two binding sites in the SLBP RBD for the histone mRNA hairpin. The first binding site (Glu129-Val158) consists of a helix-turn-helix (HTH) motif that likely recognizes the unpaired uridines in the loop of the histone hairpin and upon binding, destabilizes the first G-C base-pair at the base of the stem. The second binding site lies between residues Arg180-Pro200 which appears to recognize the second G-C basepair from the base of the stem and possibly regions flanking the stem-loop. We show that the SLBP-histone mRNA complex is regulated by threonine phosphorylation and proline isomerization in a conserved TPNK sequence that lies between the two binding sites. Threonine phosphorylation increases the affinity of SLBP for histone mRNA by slowing the off-rate for complex dissociation whereas the adjacent proline acts as a critical hinge that may orient the second binding site for formation of a stable SLBP-histone mRNA complex. The NMR and kinetic studies presented here provide a framework for understanding how SLBP recognizes histone mRNA and highlight possible structural roles of phosphorylation and proline isomerization in RNA-binding proteins in remodeling ribonucleoprotein complexes

Targeting Medium-Chain Acyl-CoA Dehydrogenase for Glioblastoma

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Mechanism of efficient double-strand break repair by a long non-coding RNA.

Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy

Regulation of DNA Double-Strand Break Repair by Non-Coding RNAs

DNA double-strand breaks (DSBs) are deleterious lesions that are generated in response to ionizing radiation or replication fork collapse that can lead to genomic instability and cancer. Eukaryotes have evolved two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ) to repair DSBs. Whereas the roles of protein-DNA interactions in HR and NHEJ have been fairly well defined, the functions of small and long non-coding RNAs and RNA-DNA hybrids in the DNA damage response is just beginning to be elucidated. This review summarizes recent discoveries on the identification of non-coding RNAs and RNA-mediated regulation of DSB repair

Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

The peptidyl-prolyl cis-trans isomerases (PPIases) that include immunophilins (cyclophilins and FKBPs) and parvulins (Pin1, Par14, Par17) participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo

The Prolyl Isomerase Pin1 Regulates mRNA Levels of Genes with Short Half-Lives by Targeting Specific RNA Binding Proteins

<div><p>The peptidyl-prolyl isomerase Pin1 is over-expressed in several cancer tissues is a potential prognostic marker in prostate cancer, and Pin1 ablation can suppress tumorigenesis in breast and prostate cancers. Pin1 can co-operate with activated ErbB2 or Ras to enhance tumorigenesis. It does so by regulating the activity of proteins that are essential for gene expression and cell proliferation. Several targets of Pin1 such as c-Myc, the Androgen Receptor, Estrogen Receptor-alpha, Cyclin D1, Cyclin E, p53, RAF kinase and NCOA3 are deregulated in cancer. At the posttranscriptional level, emerging evidence indicates that Pin1 also regulates mRNA decay of histone mRNAs, <i>GM-CSF</i>, <i>Pth</i>, and <i>TGFβ</i> mRNAs by interacting with the histone mRNA specific protein SLBP, and the ARE-binding proteins AUF1 and KSRP, respectively. To understand how Pin1 may affect mRNA abundance on a genome-wide scale in mammalian cells, we used RNAi along with DNA microarrays to identify genes whose abundance is significantly altered in response to a Pin1 knockdown. Functional scoring of differentially expressed genes showed that Pin1 gene targets control cell adhesion, leukocyte migration, the phosphatidylinositol signaling system and DNA replication. Several mRNAs whose abundance was significantly altered by Pin1 knockdown contained AU-rich element (ARE) sequences in their 3′ untranslated regions. We identified HuR and AUF1 as Pin1 interacting ARE-binding proteins <i>in vivo</i>. Pin1 was also found to stabilize all core histone mRNAs in this study, thereby validating our results from a previously published study. Statistical analysis suggests that Pin1 may target the decay of essential mRNAs that are inherently unstable and have short to medium half-lives. Thus, this study shows that an important biological role of Pin1 is to regulate mRNA abundance and stability by interacting with specific RNA-binding proteins that may play a role in cancer progression.</p></div

Pin1 acts in concert with specific RNA binding proteins to regulate the mRNA abundance of histone mRNAs and the <i>c-FOS</i> mRNA.

<p>(A) qRT-PCR analysis of histone mRNA expression in response to siRNA knockdown of Pin1, SLBP, and a double Pin1/SLBP RNAi knockdown in HEK293T cells is shown for a subset of histone mRNAs. The average change in histone mRNA levels for all five histone genes probed is shown in (B). In (C), qRT-PCR analysis of the c-FOS mRNA in response to siRNA knockdown of Pin1, HuR, AUF1, KSRP, and the double Pin1/ARE-BP RNAi knockdown in HeLa cells is shown.</p