Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product

Abstract

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called “residual” CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

“…3). Consistent with previous reports from tissue-culture systems 20,23 , U85 scaRNA and cyan fluorescent protein (CFP)-SMN continued to localize in residual bodies in coilin morphants at the same stage ( Supplementary Figs. 4b and 5).…”

“…The number of snRNPcontaining foci per nucleus in the coilin morphants was significantly decreased by a factor of 5 on average (Fig. 2b), consistent with a requirement for coilin in CB integrity 23,24 . Notably, embryos rescued by co-injection with YFP-coilin showed CBs defined by YFP-coilin protein and endogenous snRNPs (Fig.…”

“…a r t i c l e s in CB assembly 23,24 . The fact that injection of purified human snRNPs bypasses the embryonic requirement for coilin indicates that a deficit in snRNP assembly is the primarily lesion caused by coilin depletion.…”

“…Notably, depletion of coilin results in the dispersal of many CB components. Without coilin, spliceosomal factors become nucleoplasmic, and other classes of CB-localized factors (for example, SMN, fibrillarin and small Cajal body-specific RNAs (scaRNAs)) continue to self-associate in discrete 'residual bodies' [19][20][21][22][23] . Moreover, tethering of coilin to a particular nuclear site induces formation of a CB at that site 24 .…”

“…Depletion of coilin from HeLa cells by RNAi slows cell growth 20 , whereas Drosophila coilin-null mutants appear phenotypically wild type 21 . Interestingly, homozygous mouse mutants lacking 85% of the C-terminal part of the coilin coding region are significantly underrepresented among the progeny of heterozygous parents 23,29 , suggesting that coilin function may become limiting in the context of vertebrate embryogenesis. Here, we take advantage of the rapid, external development of the zebrafish Danio rerio to investigate the function of coilin and CBs.…”