Characterization of Ribonucleoprotein Complexes and Their Binding Sites on the Neurofilament Light Subunit mRNA

Rafaela Cañete-Soler,Michael L Schwartz,Yue Hua,William W Schlaepfer

doi:10.1074/jbc.273.20.12655

Rafaela Cañete-Soler, Michael L Schwartz + Show 2 more

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https://doi.org/10.1074/jbc.273.20.12655

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Abstract

Levels of neurofilament (NF) gene expression are important determinants of basic neuronal properties, but overexpression can lead to motoneuron degeneration in transgenic mice. In a companion study (Cañete-Soler, R., Schwartz, M. L., Hua, Y., and Schlaepfer, W. W. (1998) J. Biol. Chem. 273, 12650-12654), we show that levels of NF expression are regulated by altering mRNA stability and that stability determinants are present in the 3'-coding region (3'-CR) and 3'-untranslated region (3'-UTR) of the NF light subunit (NF-L) transcript. This study characterizes the ribonucleoprotein complexes that bind to the NF-L mRNA when cytoplasmic brain extracts are incubated with radioactive probes. Gel retardation assays reveal ribonucleoprotein complexes that are selectively competed with poly(C) or poly(U))/poly(A) homoribopolymers and are referred to as C-binding and U/A-binding complexes, respectively. The C-binding complex forms on the proximal 45 nucleotides of 3'-UTR, but its assembly is markedly enhanced by 23 nucleotides of flanking 3'-CR sequence. U/A-binding complexes form at multiple binding sites in the 3'-CR and 3'-UTR. A pattern of reciprocal binding suggests that the C-binding and U/A-binding complexes interact and may compete for common components or binding sites. Cross-linking studies reveal unique polypeptides in the C-binding and U/A-binding complexes. The findings provide the basis for probing mechanisms regulating NF-L mRNA stability and the relationship between NF overexpression and motoneuron degeneration in transgenic mice.

Highlights

Neurofilaments (NFs)1 are the principle constituent of the axonal cytoskeleton, so that levels of NF expression are believed to be a major determinant of axonal size
In a companion study [1], we demonstrate the presence of stability determinants in the 3Ј-coding region (3Ј-CR) and 3Ј-untranslated region (3Ј-UTR) of NF-L mRNA and have localized a stabilizing element at the junction of 3Ј-CR and 3Ј-UTR that may account for the stabilizing properties of the transcript
The present study examines the ribonucleoprotein (RNP) complexes that assemble on NF-L mRNA when nascent radioactive fragments of the transcript are incubated with cytosolic brain extracts

Summary

Introduction

Neurofilaments (NFs) are the principle constituent of the axonal cytoskeleton, so that levels of NF expression are believed to be a major determinant of axonal size. This view is supported by the simultaneous up-regulation of the light (NFL), midsized (NF-M), and heavy (NF-H) NF subunit mRNAs [2] that accompanies the enlargement and myelination of axons during postnatal development of the nervous system [3]. The extent of axonal enlargement, and presumably the levels of NF up-regulation, are determined by the nature of target cell innervations and can be reconstituted in transected nerve upon successful reinnervation of the target site [6]. The goal of the present study is to correlate biochemical with functional studies [1] and thereby develop a working model for elucidating the mechanisms that regulate the stability of the mouse NF-L transcript

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