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

Pin1 interacts with a subset of phosphorylated RNA binding proteins.

<p>Cell lysates from 293T cells were immunoprecipitated with either an anti-Pin1 or anti-hemagglutinin (HA)-specific antibody. The immunoprecipitates were resolved on a 15% SDS-PAGE and analyzed by western blotting for several ARE-BPs, Pin1, and SLBP. Five percent of the input sample was analyzed in lane 1, proteins bound to the anti-Pin1 antibody in the presence (lane 2) or absence of RNAseA (lane 3), and proteins bound to the anti-HA antibody in the absence of RNAseA in lane 4.</p

Correlation between mRNA half-lives of the target genes identified and change in mRNA abundance in the Pin1 siRNA knockdown.

<p><i>(Top)</i> In (A), the fold change in mRNA abundance for genes whose mRNA levels increase at least two fold is plotted against their half-life. In (B), the fold change in mRNA abundance for genes whose mRNA levels decrease at least two fold is plotted against their half-life. <i>(Bottom)</i> Frequency distribution of target genes that are either stabilized (left) or destabilized (right) in Pin1 siRNA treated cells as a function of mRNA half-life. Genes that show the largest fold stabilization in a Pin1 siRNA knockdown have half-lives <4 hr.</p

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 structure. Here we provide insight into the mechanism by which SLBP contacts the histone mRNA. There are two binding sites in the SLBP RNA binding domain for the histone mRNA hairpin. The first binding site (Glu129–Val158) consists of a helix–turn–helix 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 and Pro200, which appears to recognize the second G-C base pair from the base of the stem and possibly regions flanking the stem–loop structure. 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 nuclear magnetic resonance 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

Assembly of the SLIP1–SLBP Complex on Histone mRNA Requires Heterodimerization and Sequential Binding of SLBP Followed by SLIP1

The SLIP1–SLBP complex activates translation of replication-dependent histone mRNAs. In this report, we describe how the activity of the SLIP1–SLBP complex is modulated by phosphorylation and oligomerization. Biophysical characterization of the free proteins shows that whereas SLIP1 is a homodimer that does not bind RNA, human SLBP is an intrinsically disordered protein that is phosphorylated at 23 Ser/Thr sites when expressed in a eukaryotic expression system such as baculovirus. The bacterially expressed unphosphorylated SLIP1–SLBP complex forms a 2:2 high-affinity (<i>K</i>_D < 0.9 nM) heterotetramer that is also incapable of binding histone mRNA. In contrast, phosphorylated SLBP from baculovirus has a weak affinity (<i>K</i>_D ∼3 μM) for SLIP1. Sequential binding of phosphorylated SLBP to the histone mRNA stem–loop motif followed by association with SLIP1 is required to form an “active” ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to the SLIP1–SLBP heterodimer. Using alanine scanning mutagenesis, we demonstrate that the binding site on SLIP1 for SLBP lies close to the dimer interface. A single-point mutant near the SLIP1 homodimer interface abolished interaction with SLBP in vitro and reduced the abundance of histone mRNA in vivo. On the basis of these biophysical studies, we propose that oligomerization and SLBP phosphorylation may regulate the SLBP–SLIP1 complex in vivo. SLIP1 may act to sequester SLBP in vivo, protecting it from proteolytic degradation as an inactive heterotetramer, or alternatively, formation of the SLIP1–SLBP heterotetramer may facilitate removal of SLBP from the histone mRNA prior to histone mRNA degradation