Nucleolin Is a Second Component of the CD154 mRNA Stability Complex That Regulates mRNA Turnover in Activated T Cells

Abstract

CD154 (CD40L) mRNA turnover is regulated in part at the posttranscriptional level by a protein complex (termed Complex I) that binds to a highly CU-rich region of the 3′UTR. Polypyrimidine tract-binding protein (PTB) has previously been identified as a major RNA-binding protein in Complex I. Nondenaturing gel filtration of total extract from Jurkat T cells demonstrated that the CD154 mRNA-binding activity migrates as a ∼200-kDa complex, indicating the presence of multiple complex-associated proteins. We have currently undertaken a biochemical approach to further characterize Complex I and observed that it segregates over DEAE-Sepharose into two subcomplexes (termed I-L and I-U). Furthermore, nucleolin was identified as a component of both subcomplexes and was shown that it is the major RNA-binding protein in I-U. To directly demonstrate the biological significance of Complex I binding to the CD154 transcript, cytoplasm from human Jurkat cells was fractionated over a sucrose gradient and the different cellular fractions subjected to immunoprecipitation with anti-PTB and anti-nucleolin Abs. RT-PCR of the immunoprecipitated products using CD154-specific primers clearly demonstrated that nucleolin and PTB are associated with CD154 mRNA in both the ribonucleoprotein and polysome fractions. These data strongly support a model whereby nucleolin and PTB are integral to the stability of CD154 mRNA and are components of the CD154 ribonucleoprotein particle associated with actively translating ribosomes.

A Nucleolin-Binding 3′ Untranslated Region Element Stabilizes β-Globin mRNA In Vivo

“…The core structure, which comprises acidic and glycinerich domains as well as four RNA-binding domains (RBDs) (43), is extensively modified by targeted proteolysis (14,24), phosphorylation (13,54,59,60,67), ADP ribosylation (44), and methylation (46), resulting in combinatorial structural complexity that may form the basis for its observed functional heterogeneity. The four centrally positioned RBDs of nucleolin mediate its interaction with RNA both in the nucleus (32,33) and in the cytoplasm (15,56,58,69,71,72,85,86). These domains, which are structurally similar to RBDs in protein factors that regulate the stabilities and translational efficiencies of other mRNAs (22), appear to subserve a parallel spectrum of functions in nucleolin.…”

Separate cis-trans Pathways Post-transcriptionally Regulate Murine CD154 (CD40 Ligand) Expression

“…Analysis of Poly(A) Tail Length-Poly(A) tail length was measured by a ligase-mediated polyadenylation tail assay (LM-PAT) assay (31), in which the 3Ј-end of the poly(A) tail was hybridized to a primer containing oligo(dT) 16 and a GC "anchor" sequence, 5Ј-GCGAGCTCCGCGGCCGCT 16 -3Ј (Operon). Target mRNA (100 ng) was incubated with phosphorylated oligo(dT) 16 (Roche Applied Science) at 42°C in the presence of 10 units of T4 DNA ligase (Invitrogen) saturating the poly(A) tail, thereby creating an oligo(dT) copy of the poly(A) tail.…”

“…Target mRNA (100 ng) was incubated with phosphorylated oligo(dT) 16 (Roche Applied Science) at 42°C in the presence of 10 units of T4 DNA ligase (Invitrogen) saturating the poly(A) tail, thereby creating an oligo(dT) copy of the poly(A) tail. At 42°C, the 3Ј-end of the poly(A) tail remained largely unpaired due to unfavorable hybridization conditions.…”

“…Deletions that eliminated PTB binding to the CD154 3Ј-UTR decreased the stability of CD154 mRNA seen in D1.1 cells. Subsequent work suggested that the interaction of PTB and nucleolin with this region of the CD154 3Ј-UTR correlated with the increased mRNA stability (16). Thus, the stabilization of CD154 mRNA that occurs with prolonged T cell activation involves the differential effects of PTB splice isoforms as well as the interaction of PTB with other trans-acting factors.…”

mRNA stability control: a clandestine force in normal and malignant hematopoiesis

“…Other proteins implicated in RNA stabilization include RNPC1, 90 the poly (C)-binding protein aCP1, 91 and the ARE-binding proteins NF90, 92,93 and nucleolin. [94][95][96][97] The aCP-binding proteins are important in stabilizing aglobin mRNA and have been reviewed elsewhere. 98 Nucleolin has been investigated extensively for its roles in transcription, splicing and nucleolar function (reviewed by Ginisty et al 99 ) but only a handful of papers have examined it as a regulator of mRNA turnover.…”