The NF45/NF90 Heterodimer Contributes to the Biogenesis of 60S Ribosomal Subunits and Influences Nucleolar Morphology

Abstract

dThe interleukin enhancer binding factors ILF2 (NF45) and ILF3 (NF90/NF110) have been implicated in various cellular pathways, such as transcription, microRNA (miRNA) processing, DNA repair, and translation, in mammalian cells. Using tandem affinity purification, we identified human NF45 and NF90 as components of precursors to 60S (pre-60S) ribosomal subunits. NF45 and NF90 are enriched in nucleoli and cosediment with pre-60S ribosomal particles in density gradient analysis. We show that association of the NF45/NF90 heterodimer with pre-60S ribosomal particles requires the double-stranded RNA binding domains of NF90, while depletion of NF45 and NF90 by RNA interference leads to a defect in 60S biogenesis. Nucleoli of cells depleted of NF45 and NF90 have altered morphology and display a characteristic spherical shape. These effects are not due to impaired rRNA transcription or processing of the precursors to 28S rRNA. Consistent with a role of the NF45/NF90 heterodimer in nucleolar steps of 60S subunit biogenesis, downregulation of NF45 and NF90 leads to a p53 response, accompanied by induction of the cyclin-dependent kinase inhibitor p21/CIP1, which can be counteracted by depletion of RPL11. Together, these data indicate that NF45 and NF90 are novel higher-eukaryote-specific factors required for the maturation of 60S ribosomal subunits. T he nuclear factors NF45 and NF90 (NFAR-1, DRBP76, MPP4, and TCP80) were originally discovered as a heterodimeric complex binding to the interleukin-2 (IL-2) promoter (1, 2) and are also referred to as interleukin enhancer-binding factors 2 (ILF2) and 3 (ILF3), respectively (3). While NF90 is vertebrate specific, NF45 is found throughout metazoans.In mammals, the NF45/NF90 complex is widely expressed across tissues (4). Over recent years, NF45/NF90 has been implicated in a great variety of biological processes. Apart from regulation of transcription (5-7), the heterodimer has also been linked to numerous other pathways, such as DNA damage response (8, 9), mRNA metabolism (10, 11), microRNA (miRNA) biogenesis (12), and viral infection (13-17). NF90 knockout mice display severe defects in skeletal muscle formation leading to respiratory failure soon after birth (18), indicating an essential role of NF90 function in vertebrate development.Both NF45 and NF90 possess an N-terminal "domain associated with zinc fingers" (DZF) that is found only in metazoan proteins. Recent structural analysis revealed that the DZF domains of NF45 and NF90 resemble template-free nucleotidyltransferases and mediate their heterodimerization through a structurally conserved interface (19). In addition to the DZF domain, NF90 possesses two double-stranded RNA binding domains (dsRBDs) in the C-terminal region (2, 20) that confer binding to highly structured RNAs (21-23).NF90 is expressed from at least five alternatively spliced mRNAs that all encode the DZF and dsRBDs. Some of the splice variants generate C-terminally extended protein isoforms referred to as NF110 (NFAR-2) (24, 25), which also interact with ...

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

“…3a) [27]. Two of these factors have previous links to nucleolar activity, as ILF3 (also known as NF90) was previously shown to associate with pre-60S ribosomal particles in the nucleolus and knockdown of ILF3 gives defects in rRNA biogenesis [28,30], and LIN28B has been shown to repress let-7 processing by sequestering pri-let-7 in the nucleolus [31]. In this region, multiple sites of ILF3 and SSB enrichment flank a more specific region enriched in LIN28B eCLIP (Fig.…”

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

“…3a) [26]. Two of these factors have previous links to nucleolar activity, as ILF3 (also known as NF90) was previously shown to associate with pre-60S ribosomal particles in the nucleolus and knockdown of ILF3 gives defects in rRNA biogenesis [27,29], and LIN28B has been shown to repress let-7 processing by sequestering pri-let-7 in the nucleolus [30]. In this region, multiple sites of ILF3 and SSB enrichment flank a more specific region enriched in LIN28B eCLIP (Fig 3e, Sup.…”

MiR-590-5p inhibits colorectal cancer angiogenesis and metastasis by regulating nuclear factor 90/vascular endothelial growth factor A axis

“…14 The two most prominent protein isoforms are termed NF90 (also known as DRBP76 and NFAR1) and NF110 (also known as ILF3, NFAR2, and TCP110), and have apparent molecular masses of 90 and 110 kDa, respectively. 14 Although both NF90 and NF110 are homologous in the N-terminal and central regions, their C-terminal are completely different. 14 NF90 was originally purified as a DNA-binding complex regulating the interleukin-2 (IL-2) promoter.…”

“…14 Although both NF90 and NF110 are homologous in the N-terminal and central regions, their C-terminal are completely different. 14 NF90 was originally purified as a DNA-binding complex regulating the interleukin-2 (IL-2) promoter. 15 However, its function is not restricted to T cells because NF90 was found to regulate a number of other mRNAs.…”