The anterior pituitary controls key biological processes, including growth, metabolism, reproduction and stress responses through distinct cell types that each secrete specific hormones. The anterior pituitary cells show a remarkable level of cell type plasticity that mediates the shifts in hormone producing cell populations that are required to meet organismal needs. The molecular mechanisms underlying pituitary cell plasticity are not well understood. Recent work has implicated the pituitary stem cell populations and specifically, the mRNA binding proteins of the Musashi family in control of pituitary cell type identity. In this study we have identified the target mRNAs that mediate Musashi function in the adult mouse pituitary and demonstrate the requirement for Musashi function in vivo. Using Musashi RNA immunoprecipitation, we identify a cohort of 1184 mRNAs that show specific Musashi binding. Identified Musashi targets include the Gnrhr mRNA, which encodes the gonadotropin releasing hormone receptor (GnRHR), and the Fshb mRNA, encoding follicle-stimulating hormone (FSH). Reporter assays reveal that Musashi functions to exert repression of translation of the Fshb mRNA, in addition to the previously observed repression of the Gnrhr mRNA. Importantly, mice engineered to lack Musashi in gonadotropes demonstrate a failure to repress translation of the endogenous Gnrhr and Fshb mRNAs during the estrous cycle and display a significant heterogeneity in litter sizes. The range of identified target mRNAs suggests that, in addition to these key gonadotrope proteins, Musashi may exert broad regulatory control over the pituitary proteome in a cell-type specific manner.

The adipokine leptin regulates energy homeostasis through ubiquitously expressed leptin receptors. Leptin has a number of major signaling targets in the brain, including cells of the anterior pituitary (AP). We have previously reported that mice lacking leptin receptors in AP somatotropes display growth hormone (GH) deficiency, metabolic dysfunction, and adult-onset obesity. Among other targets, leptin signaling promotes increased levels of the pituitary transcription factor POU1F1, which in turn regulates the specification of somatotrope, lactotrope, and thyrotrope cell lineages within the AP. Leptin’s mechanism of action on somatotropes is sex dependent, with females demonstrating posttranscriptional control of Pou1f1 messenger RNA (mRNA) translation. Here, we report that the stem cell marker and mRNA translational control protein, Musashi1, exerts repression of the Pou1f1 mRNA. In female somatotropes, Msi1 mRNA and protein levels are increased in the mouse model that lacks leptin signaling (Gh-CRE Lepr-null), coincident with lack of POU1f1 protein, despite normal levels of Pou1f1 mRNA. Single-cell RNA sequencing of pituitary cells from control female animals indicates that both Msi1 and Pou1f1 mRNAs are expressed in Gh-expressing somatotropes, and immunocytochemistry confirms that Musashi1 protein is present in the somatotrope cell population. We demonstrate that Musashi interacts directly with the Pou1f1 mRNA 3′ untranslated region and exerts translational repression of a Pou1f1 mRNA translation reporter in a leptin-sensitive manner. Musashi immunoprecipitation from whole pituitary reveals coassociated Pou1f1 mRNA. These findings suggest a mechanism in which leptin stimulation is required to reverse Musashi-mediated Pou1f1 mRNA translational control to coordinate AP somatotrope function with metabolic status.

after MCAO (Fig. 7B and Supplementary Fig. 1B) although the pAkt concentration was unaffected 48 h after the MCAO. Therefore, we conclude that activation of GPR35 on monocyte/macrophages by its ligand pamoic acid reprograms these cell types into neuroprotective pathways. Method Mice. Male Swiss albino mice (8-12 weeks) were collected from the North South University (NSU) animal house and were maintained under standard environmental conditions (temperature 23.0 ± 2.0 °C, relative humidity: 55-65% and 12 h light and dark cycle). All experiments were carried out according to the institutional guideline and were approved by the NSU Institutional Animal Care and Use Committee (IACUC).

The pituitary gland controls responses to changing physiological requirements during growth, pregnancy, puberty, and stress. Synthesis and secretion of appropriate pituitary hormones in response to changing organismal demands is mediated through a remarkable capacity for cellular plasticity of the distinct hormone-producing cells. The mechanisms governing pituitary cellular plasticity are not fully understood. We hypothesize that plasticity in the adult tissue is mediated through similar mechanisms to those that occur during embryonic and postnatal pituitary development. The Prophet of Pit1(Prop1), a paired-like homeodomain transcription factor, is the earliest pituitary-specific transcription factor and is highly expressed in pituitary progenitor cells where it controls lineage commitment to the distinct hormone-producing cell types. Our recent work has identified post-transcriptional regulation of pituitary gene expression in the adult tissue via the stem cell determinant, mRNA binding protein, Musashi. The full-length mouse Prop1 (mProp1) mRNA 3' untranslated region (UTR) contains 24 consensus binding elements for Musashi (MBEs) and demonstrates Musashi1-dependent translational activation in vitro. Specifically, the terminal mProp1 3' UTR MBE directs 40% of Musashi1-mediated translation activation, and mutational disruption of this terminal MBE abolishes all Musashi1-dependent translational activation. Immunoprecipitation of Musashi-mRNA complexes from adult mouse pituitaries confirmed Musashi1-mProp1 mRNA association in adult tissue. The gene sequence data for the human Prop1 (hPROP1) mRNA 3' UTR is incomplete in the Ensembl and RefSeq databases. Using published RNA sequencing datasets, we identified an expressed full-length hPROP1 mRNA 3' UTR of 6996 nucleotides with 54 predicted consensus MBEs, suggesting that the human PROP1 mRNA, like the murine Prop1 mRNA, may be subject to Musashi-dependent translational control. Our findings are consistent with the hypothesis that the Musashi protein exerts evolutionarily conserved control of Prop1 mRNA translation to facilitate plasticity of anterior pituitary function. Presentation: Saturday, June 11, 2022 1:31 p.m. - 1:36 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The Musashi RNA-binding protein functions as a gatekeeper of cell maturation and maintains stem cell plasticity by regulating the translation of target mRNAs. The adult anterior pituitary tissue expresses a high level of Musashi and also demonstatres a high level of cell plasticity, indicating a Musashi-dependent function in maintaining endocrine homeostasis in the anterior pituitary. Towards understanding the mechanism(s) by which Musashi functions to control cell plasticity, we have identified co-associated proteins necessary for Musashi function. The two Musashi isoforms (Musashi1 and Musashi2) are conserved across species and are structurally characterized by two N-terminal RNA-recognition motifs (RRMs, that associate with specific sequences in the 3' untranslated region of mRNAs targets) and a disordered C-terminus. Musashi uses these domains to selectively repress, or to activate, mRNA translation in a manner specific to target mRNA and cell context. Both Musashi isoforms contain two C-terminal regulatory serine residues that require phosphorylation to enable mRNA translational activation. However, the Musashi proteins do not have inherent mRNA translation function and utilize co-associated protein complexes to mediate mRNA translation. We have previously identified Musashi1 binding protein partners through mass spectrometry. Here, we have identified the protein interactions that are required for Musashi1-dependent translational activation. Knockdown of target protein levels in Xenopus laevis oocytes indicated that the PABP4, CELF2, LSM14A/B, ELAVL1, ELAVL2, ELAVL4, and PUM1 proteins are required for Musashi target mRNA translational activation. We further determined that the effect of LSM14A/B dual knockdown was in fact specific to LSM14B alone. LSM14B is an RNA-binding protein that is required for RNA granule formation, RNA transcript metabolism, and mRNA translation. Studies in mouse oocytes have shown that LSM14B is required to maintain transcript levels of the Musashi1 target Cyclin B1 (Ccnb1) mRNA prior to mRNA translation. In the adult murine pituitary, LSM14B is ubiquitously expressed in both hormone-producing cells and in stem cells as indicated through single cell RNA sequencing. These findings indicate that LSM14B and Musashi1 may function cooperatively to regulate pituitary hormone production and cell differentiation in response to changes in physiological demand. Presentation: Saturday, June 11, 2022 11:45 a.m. - 12:00 p.m.