A biosensor for MAPK-dependent Lin28 signaling

Abstract

Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the posttranscriptional level, and can undergo mitogen-activated protein kinase (MAPK)–mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR RNA-binding protein (TRBP). However, molecular and spatiotemporal details of this critical control mechanism remained unknown. In this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop biosensors to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support coassociation and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative Erk-target serine 152, as well as Lin28a serine 200 phosphorylation, in mediating the increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop fluorescence resonance energy transfer (FRET)-based sensors for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate the response of bimolecular and unimolecular FRET sensors to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation.

A Fyn - specific biosensor reveals localized, pulsatile kinase activity and spatially regulated signaling crosstalk

“…Calculations used are consistent with other literature reports on intermolecular FRET measurements, like the Fiji plugin “FRET analyzer” 114 and another very recent report 115 .…”

Growth-suppressor microRNAs mediate synaptic overgrowth and behavioral deficits in Fragile X mental retardation protein deficiency

“… 81 , 82 Neuronal activity induced by EE elevates trophic factors, including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and MAPK activity, which have been associated with induction of the Lin28 RNA binding protein and consequent reduction in mature let-7 family miRNA levels. 44 , 60 , 83 , 84 , 85 Short-term EE (or novel environment exposure) allows the study of primary responses, such as immediate-early genes or post-transcriptional impacts, rather than secondary responses which can be investigated with long-term EE. 86 , 87 , 88 , 89 We exposed adult WT and Fmr1 KO mice (male, P60 age-matched) to either their acclimated home cage (sham) or to short-term EE in a cage containing both novel objects and mice for 2 h, followed by immediate isolation of hippocampi and qRT-PCR.…”

“…Work from our laboratory and others identified a MAPK-dependent mechanism for signal-induced stabilization and upregulation of Lin28a in differentiated cells, through Lin28a phosphorylation and binding to phosphorylated TAR RNA binding protein (TRBP), which result in consequent reduced levels of Lin28-targeted let-7 family miRNAs. 44 , 60 , 61 , 83 MAPK pathway dysregulation has been reported in FXS, with elevated levels of phospho-extracellular signal-related kinase 1 and 2 (pERK1/2) in brain and peripheral tissues from Fmr1 KO mice and FXS patients. 8 , 90 , 91 , 92 Given these observations, we hypothesized that hyperactive MAPK signaling might participate in the reductions in let-7 family miRNAs associated with loss of FMRP by induction of the Lin28a RNA binding protein.…”

Regulation by noncoding RNAs of local translation, injury responses, and pain in the peripheral nervous system

“…One underlying mechanism involves the impact of these kinase cascades on microRNA (miRNA) biogenesis and miRNA-mediated repression ( Amen et al, 2017 ; Y.-W. A. Huang et al, 2012 , Oldach et al, 2018 , Totary-Jain et al, 2013 ; P. Ye et al, 2015 ).…”