Brain Long Noncoding RNAs: Multitask Regulators of Neuronal Differentiation and Function.

The extraordinary cellular diversity and the complex connections established within different cells types render the nervous system of vertebrates one of the most sophisticated tissues found in living organisms. Such complexity is ensured by numerous regulatory mechanisms that provide tight spatiotemporal control, robustness and reliability. While the unusual abundance of long noncoding RNAs (lncRNAs) in nervous tissues was traditionally puzzling, it is becoming clear that these molecules have genuine regulatory functions in the brain and they are essential for neuronal physiology. The canonical view of RNA as predominantly a 'coding molecule' has been largely surpassed, together with the conception that lncRNAs only represent 'waste material' produced by cells as a side effect of pervasive transcription. Here we review a growing body of evidence showing that lncRNAs play key roles in several regulatory mechanisms of neurons and other brain cells. In particular, neuronal lncRNAs are crucial for orchestrating neurogenesis, for tuning neuronal differentiation and for the exact calibration of neuronal excitability. Moreover, their diversity and the association to neurodegenerative diseases render them particularly interesting as putative biomarkers for brain disease. Overall, we foresee that in the future a more systematic scrutiny of lncRNA functions will be instrumental for an exhaustive understanding of neuronal pathophysiology.

The codon sequences predict protein lifetimes and other parameters of the protein life cycle in the mouse brain.

The homeostasis of the proteome depends on the tight regulation of the mRNA and protein abundances, of the translation rates, and of the protein lifetimes. Results from several studies on prokaryotes or eukaryotic cell cultures have suggested that protein homeostasis is connected to, and perhaps regulated by, the protein and the codon sequences. However, this has been little investigated for mammals in vivo. Moreover, the link between the coding sequences and one critical parameter, the protein lifetime, has remained largely unexplored, both in vivo and in vitro. We tested this in the mouse brain, and found that the percentages of amino acids and codons in the sequences could predict all of the homeostasis parameters with a precision approaching experimental measurements. A key predictive element was the wobble nucleotide. G-/C-ending codons correlated with higher protein lifetimes, protein abundances, mRNA abundances and translation rates than A-/U-ending codons. Modifying the proportions of G-/C-ending codons could tune these parameters in cell cultures, in a proof-of-principle experiment. We suggest that the coding sequences are strongly linked to protein homeostasis in vivo, albeit it still remains to be determined whether this relation is causal in nature.

Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions.

The turnover of brain proteins is critical for organism survival, and its perturbations are linked to pathology. Nevertheless, protein lifetimes have been difficult to obtain in vivo. They are readily measured in vitro by feeding cells with isotopically labeled amino acids, followed by mass spectrometry analyses. In vivo proteins are generated from at least two sources: labeled amino acids from the diet, and non-labeled amino acids from the degradation of pre-existing proteins. This renders measurements difficult. Here we solved this problem rigorously with a workflow that combines mouse in vivo isotopic labeling, mass spectrometry, and mathematical modeling. We also established several independent approaches to test and validate the results. This enabled us to measure the accurate lifetimes of ~3500 brain proteins. The high precision of our data provided a large set of biologically significant observations, including pathway-, organelle-, organ-, or cell-specific effects, along with a comprehensive catalog of extremely long-lived proteins (ELLPs).

miR-99a/100~125b tricistrons regulate hematopoietic stem and progenitor cell homeostasis by shifting the balance between TGFß and Wnt signaling.

Although regulation of stem cell homeostasis by microRNAs (miRNAs) is well studied, it is unclear how individual miRNAs genomically encoded within an organized polycistron can interact to induce an integrated phenotype. miR-99a/100, let-7, and miR-125b paralogs are encoded in two tricistrons on human chromosomes 11 and 21. They are highly expressed in hematopoietic stem cells (HSCs) and acute megakaryoblastic leukemia (AMKL), an aggressive form of leukemia with poor prognosis. Here, we show that miR-99a/100∼125b tricistrons are transcribed as a polycistronic message transactivated by the homeobox transcription factor HOXA10. Integrative analysis of global gene expression profiling, miRNA target prediction, and pathway architecture revealed that miR-99a/100, let-7, and miR-125b functionally converge at the combinatorial block of the transforming growth factor ß (TGFß) pathway by targeting four receptor subunits and two SMAD signaling transducers. In addition, down-regulation of tumor suppressor genes adenomatous polyposis coli (APC)/APC2 stabilizes active ß-catenin and enhances Wnt signaling. By switching the balance between Wnt and TGFß signaling, the concerted action of these tricistronic miRNAs promoted sustained expansion of murine and human HSCs in vitro or in vivo while favoring megakaryocytic differentiation. Hence, our study explains the high phylogenetic conservation of the miR-99a/100∼125b tricistrons controlling stem cell homeostasis, the deregulation of which contributes to the development of AMKL.

Expression of a2, a5 and a6 subunits of integrin in de-differentiated NIH3T3 cells by cell-free extract of embryonic stem cells.

Generation of patient specific stem cells is among the ultimate goals in regenerative medicine. Such a cell needs to be functional when it transplants. Interaction between the matrix proteins and integrin adjust many cells' function such as adhesion, migration, cell cycle and self renewal in stem cells. In this study, NIH3T3 cells were dedifferentiated by mouse Embryonic Stem Cell (mESC) extract. The expression of pluripotency markers as well as a2, a5 and a6 integrin subunits were determined. NIH3T3 cells treated with mESC extract showed noticeable changes in cell morphology as early as day 2 post-treatment forming colonies similar to typical mESC morphology by day 8, after three passages. Alkaline phosphatase (ALP) assay and immunocytochemistry staining were performed for the induced reprogrammed cells. The results indicated that these colonies showed the ALP activity and they express Sox2 and Nanog. RT-PCR revealed that the colonies also express Oct3/4. NIH3T3 cells, ESC and reprogrammed cells expressed a2 integrin. a5 integrin expression was greatest in reprogrammed cells followed by the expression of this integrin in NIH3T3 which in turn was more than in ESC. a6A integrin was expressed in NIH3T3 cells while a6B integrin was expressed in ESC and in very low quantity was expressed in reprogrammed cells. These data provide evidence for both the generation of ES like cells from differentiated somatic cells and the expression profile of integrins after de-differentiation by mESC extract.