Post-transcriptional mechanisms controlling neurogenesis and direct neuronal reprogramming

Abstract

Neurogenesis is a tightly regulated process in time and space both in the developing embryo and in adult neurogenic niches. A drastic change in the transcriptome and proteome of radial glial cells or neural stem cells towards the neuronal state is achieved due to sophisticated mechanisms of epigenetic, transcriptional, and post-transcriptional regulation. Understanding these neurogenic mechanisms is of major importance, not only for shedding light on very complex and crucial developmental processes, but also for the identification of putative reprogramming factors, that harbor hierarchically central regulatory roles in the course of neurogenesis and bare thus the capacity to drive direct reprogramming towards the neuronal fate. The major transcriptional programs that orchestrate the neurogenic process have been the focus of research for many years and key neurogenic transcription factors, as well as repressor complexes, have been identified and employed in direct reprogramming protocols to convert non-neuronal cells, into functional neurons. The post-transcriptional regulation of gene expression during nervous system development has emerged as another important and intricate regulatory layer, strongly contributing to the complexity of the mechanisms controlling neurogenesis and neuronal function. In particular, recent advances are highlighting the importance of specific RNA binding proteins that control major steps of mRNA life cycle during neurogenesis, such as alternative splicing, polyadenylation, stability, and translation. Apart from the RNA binding proteins, microRNAs, a class of small non-coding RNAs that block the translation of their target mRNAs, have also been shown to play crucial roles in all the stages of the neurogenic process, from neural stem/progenitor cell proliferation, neuronal differentiation and migration, to functional maturation. Here, we provide an overview of the most prominent post-transcriptional mechanisms mediated by RNA binding proteins and microRNAs during the neurogenic process, giving particular emphasis on the interplay of specific RNA binding proteins with neurogenic microRNAs. Taking under consideration that the molecular mechanisms of neurogenesis exert high similarity to the ones driving direct neuronal reprogramming, we also discuss the current advances in in vitro and in vivo direct neuronal reprogramming approaches that have employed microRNAs or RNA binding proteins as reprogramming factors, highlighting the so far known mechanisms of their reprogramming action.

Figure 1

Post-transcriptional mechanisms mediated by RBPs and miRNAs orchestrating neurogenesis. Post-transcriptional mechanisms controlling neurogenesis that are discussed in the text, giving particular emphasis on the role of neurogenic miRNAs in regulating RBPs expressed in neural stem/progenitor cells. Early in the course of neurogenesis the neurogenic miRNAs that are gradually upregulated, miR-124, miR-9, miR-128, miR-125, and miR-137 target for downregulation RBPs that inhibit the expression of neuronal specific mRNAs by (a) alternative splicing (AS) or alternative splicing coupled with nonsense mediated decay (AS-NMD), such as Ptbp1, (b) mRNA decay mechanisms, such as Zfp36l1 and Zfp36, (c) nonsense mediated decay (NMD), such as Upf1 and Upf3b and the kinase Smg1 that phosphorylates UPF1 and (d) translation inhibition, such as Msi. Through these actions, these neurogenic miRNAs indirectly de-repress numerous neurogenic mRNAs, such as Ptbp2, the neuronal RBPs Elavl2/3/4, Nova1/2, Rbfox1/2 and other neuronal-specific mRNAs related to axonal growth, migration, and differentiation. As discussed in the text, miR-137, apart from directly targeting Msi, also competes with MSI in directly targeting stemness-related mRNAs. Later in neurogenesis, PTBP2 levels decline (shown in lighter blue color) allowing for adult-specific alternative splicing to occur. In parallel, the increase of RBFOX3 in synergy with other neuronal specific RBPs promotes the upregulation of neuronal maturation mRNAs related to synaptic transmission, membrane excitation, cytoskeleton, and plasticity by post-transcriptional mechanisms, including (a) stabilization, (b) alternative splicing, (c) promotion of translation and (d) transport and control of local translation. Created with BioRender.com. ANKG: Ankyrin G; AREs: AU-rich elements; AS: alternative splicing; AS-NMD: alternative splicing coupled with nonsense mediated decay; ATP2B: ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 2; BAK: BCL2 antagonist/killer 1; BDNF: brain-derived neurotrophic factor; CAMK2A: calcium/calmodulin-dependent protein kinase II alpha; EGFR: epidermal growth factor receptor; ELAVL2/3/4: embryonic lethal abnormal vision-like 2/3/4; GABRG2: gamma-aminobutyric acid type A receptor subunit gamma2; GAP-43: growth associated protein 43; GPHN: gephyrin; GRIN1: glutamate ionotropic receptor NMDA type subunit 1; MSI: musashi; nELAVLs: neuronal ELAVLs; NOVA1/2: neuro-oncological ventral antigen 1/2; NRAS: Neuroblastoma Ras proto-oncogene; NRN1: neuritin 1; NTRK2: neurotrophic receptor tyrosine kinase 2; NUMB: NUMB endocytic adaptor protein; PBX1: pre B cell leukemia homeobox 1; PDGFRA: platelet-derived growth factor receptor alpha; PSD-95: postsynaptic density protein 95; PTBP1/2: polypyrimidine tract binding protein 1/2; RBFOX1/2/3: RNA binding fox-1 homolog 1/2/3; SATB1: Special AT-Rich Sequence Binding Protein 1; SMAD7: SMAD family member 7; SMG1: SMG1 nonsense mediated mRNA decay associated PI3K related kinase; SP1: SP1 transcription factor; UPF1: UPF1 RNA helicase and ATPase; UPF3B: UPF3B regulator of nonsense mediated mRNA decay; VAMP: vesicle-associated membrane protein; ZFP36: ZFP36 ring finger protein; ZFP36L1: ZFP36 ring finger protein like 1.

Figure 2

Mechanisms controlling direct reprogramming of non-neuronal cells to iNs in strategies that overexpress the neurogenic miRNAs, miR-9/9* and miR-124 or downregulate the RBPs, PTBP1, and PTBP2. Schematic presentation of the intricate mechanisms that orchestrate the direct reprogramming of astrocytes or fibroblasts to iNs after the overexpression of miR-124 alone or in combination with miR-9/9* or after the repression of PTBP1 either alone or sequentially with PTBP2. Emphasis is given on the different levels of gene expression regulation that are affected by these reprogramming approaches. The PTBP1-REST-miR-124 circuitry initiated by Ptbp1 repression and the PTBP2-BRN2-miR-9 loop initiated by the following Ptbp2 downregulation are highlighted with magenta and green arrows respectively (the dashed arrow in BRN2 repression by PTBP2 indicates an unknown direct or indirect mechanism). The positive regulation of Ptbp2 by miR-124 in synergy with ELAVL3 is also portrayed. On the other hand, overexpression of miR-124 alone or along with miR-9/9*, cooperatively downregulates core components of REST complex (Rest, CoRest, Scp1) and subsequently de-repress neuronal specific genes at the transcriptional level. These miRNAs also act in synergy to downregulate the RBPs implicated in mRNA decay, Zfp36l1, and Zfp36, promoting the stabilization of ARE-containing neuronal mRNAs at the post-transcriptional level. Additionally, miR-9/9*-124 jointly target the BAF component Baf53a reinforcing the BAF53a to BAF53b switch in the neuronal-specific nBAF chromatin remodeling complex and induce the opening of closed chromatin containing neuronal gene loci. Finally, miR-124 directly, or along with miR-9 indirectly, targets the epigenetic factor, Ezh2, which is a negative regulator of neurogenesis, as it stabilizes REST protein and also participates in the PRC2 repressor complex. Created with BioRender.com. ARE: AU-rich element; BAF53a/b: BRG1-associated factor 53 a/b; BRN2: brain-specific homeobox/POU domain protein 2 (also known as POU3F2); CoREST: REST Corepressor 1 (also known as RCOR1); ELAVL3: embryonic lethal abnormal vision-like 3; EZH2: enhancer of zeste 2 polycomb repressive complex 2 subunit; KD: knockdown; PTBP1/2: polypyrimidine tract binding protein 1/2; REST: RE1 Silencing Transcription Factor; SCP1: CTD small phosphatase 1 (also known as CTDSP1); USP14: ubiquitin specific peptidase 14; ZFP36: ZFP36 ring finger protein; ZFP36L1: ZFP36 ring finger protein like 1.