Targeting SMN to Cajal bodies and nuclear gems during neuritogenesis

Abstract

Neurite outgrowth is a central feature of neuronal differentiation. PC12 cells are a good model system for studying the peripheral nervous system and the outgrowth of neurites. In addition to the dramatic changes observed in the cytoplasm, neuronal differentiation is also accompanied by striking changes in nuclear morphology. The large and sustained increase in nuclear transcription during neuronal differentiation requires synthesis of a large number of factors involved in pre-mRNA processing. We show that the number and composition of the nuclear subdomains called Cajal bodies and gems changes during the course of N-ras-induced neuritogenesis in the PC12-derived cell line UR61. The Cajal bodies found in undifferentiated cells are largely devoid of the survival of motor neurons (SMN) protein product. As cells shift to a differentiated state, SMN is not only globally upregulated, but is progressively recruited to Cajal bodies. Additional SMN foci (also known as Gemini bodies, gems) can also be detected. Using dual-immunogold labeling electron microscopy and mouse embryonic fibroblasts lacking the coilin protein, we show that gems clearly represent a distinct category of nuclear body.

Fibroblast growth factor–2 regulates the stability of nuclear bodies

“…3B). No change in the frequency distribution between SMN/empty vector and SMN/FGF-2 18 could be observed, as these proteins did not interact. Importantly, cotransfection with FGF-2 23 revealed a significant left shift between the distributions (Fig.…”

“…Dimerization of SMN with the SMN28-294 mutant is not affected because the first self-association domain is encoded by exon 2b (amino acid residues 52-91) (28) and the second domain is in the YG-box region encoded by exon 7 (31). Concomitant with the overexpression of SMN, the average number of SMNpositive structures increased from the endogenous level of 1.8 23 showed a significant difference of the frequency distribution compared with the SMN1-294/empty vector control, whereas co-transfection with SMN1-294 and the FGF-2 18 isoform did not change the frequency distribution. As additional controls, cells were transfected with pSMN28 -294-EGFP lacking the FGF-2 23 interaction domain in the N terminus.…”

“…No difference from the SMN28 -294/empty vector control was detected. Three independent experiments were performed per condition with the following numbers of measured cells: SMN1-294/empty vector n ϭ 619; SMN1-294/FGF-2 18 , n ϭ 377; SMN1-294/FGF-2 23 , n ϭ 636; SMN28 -294/empty vector, n ϭ 557; SMN28 -294/FGF-2 18 , n ϭ 501; and SMN28 -294/FGF-2 23 , n ϭ 502. Error bars represent standard errors of the means.…”

“…SMN contains a motif encoded by exon 2b that is responsible for targeting to Cajal bodies (17). In most cell lines, Cajal bodies and gems co-localize (5), but gems represent a distinct class of nuclear substructures (11,16,18). In motoneurons, SMN-positive foci are more frequent in adult than in fetal stages.…”

“…This growth factor is expressed as different isoforms that are translated from one mRNA species. The 18 kDa isoform (FGF-2 18 ) is generated after translation initiation at a conventional AUG start codon, whereas upstream alternative CUG start codons are used for expression of the high-molecular weight isoforms (hmw) 21 and 23 kDa FGF-2 (rat FGF-2 21 and FGF-2 23 ). FGF-2 isoforms are differentially regulated during development, after pharmacological treatment, and after nerve injury (24), indicating different biological roles for the individual isoforms.…”