Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis

Abstract

0 3 a r t i c l e sIn eukaryotes, organelles delimited by lipid bilayers support the organization of cellular functions, as exemplified by the segregation of chromatin into the cell nucleus. However, many subcellular compartments-nucleoli, Cajal and PML bodies, polar granules, stress granules, P bodies and others-lack membranes. How these structures contribute to cellular physiology is largely unknown 1 . The Cajal body (CB) is a 0.5-to 1-µm nuclear compartment initially described by Ramon y Cajal in silver-stained sections of vertebrate cerebral cortex 2 . Because the CB is conserved in evolution and is uniquely marked by the protein coilin, it has served as a general model for subnuclear structure and function 1,2 . Numerous factors essential for pre-mRNA splicing, histone mRNA 3′-end processing, telomere maintenance and rRNA processing are concentrated in CBs. Yet CB components exchange rapidly with the nucleoplasm, where most of these processes occur 3 . The challenge, therefore, has been to identify the function of CBs.Despite the localization of splicing machinery in CBs, they are not the sites of pre-mRNA splicing 4,5 . Splicing occurs throughout the nucleus and is catalyzed by the spliceosome, a macromolecular complex formed on pre-mRNA from five essential snRNPs 6 . Each spliceosomal snRNP comprises a unique 100-to 200-nucleotide snRNA (U1, U2, U4, U5 and U6) that is post-transcriptionally modified and associated with a number of snRNP-specific proteins 6,7 . Several roles for CBs in spliceosomal snRNP biogenesis have been proposed based on the localization of specific steps in snRNP assembly in the CBs of cultured cell lines. After transcription and export to the cytoplasm, U1, U2, U4 and U5 snRNAs receive a heteroheptameric ring of Sm proteins assembled by the SMN complex; subsequently, their 5′ ends are hypermethylated 8 . The Sm ring and trimethylguanosine (TMG) cap are signals for snRNP reimport into the nucleus, where these still-immature core snRNPs first concentrate in CBs 9-12 . CBs contain scaRNAs, which then guide site-specific modification of the snRNAs 13 . Importantly, intermediates in the final snRNP maturation steps, reflecting RNA structural rearrangements and the recruitment of snRNP-specific proteins, are highly concentrated in CBs 14-17 . These observations have led to the proposal that CBs are the sites of snRNP assembly, but an essential role for CBs in this process has not been shown.The coilin protein may hold the key to CB function. Immunoelectron microscopy studies of CBs reveal thread-like, coilin-positive aggregates, suggesting that coilin is integral to CB structure 2 . Consistent with this, coilin resides longer in CBs than any other examined component in vivo 3,18 . 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' ...

Coilin: The first 25 years

“…17,18 In zebrafish embryos, coilin depletion through morpholino injection was lethal within the first 24 hours of development and was accompanied by reduced levels of snRNPs and spliced mRNAs. 19 Fish embryos were rescued upon injection of spliceosomal small nuclear ribonucleoprotein particles (snRNPs), which suggests that coilin's essential function in embryos is to promote macromolecular assembly of snRNPs. 19 This conclusion agrees with early findings that snRNAs and snRNP proteins are concentrated in CBs.…”

“…19 Fish embryos were rescued upon injection of spliceosomal small nuclear ribonucleoprotein particles (snRNPs), which suggests that coilin's essential function in embryos is to promote macromolecular assembly of snRNPs. 19 This conclusion agrees with early findings that snRNAs and snRNP proteins are concentrated in CBs. 20,21 Spliceosomal snRNPs are assembled and recycled in CBs.…”

“…A clear example is zebrafish, where embryos depleted of coilin and lacking CBs are unable to complete embryogenesis, display splicing defects and reduced numbers of mature snRNPs. 19,35 Because zebrafish embryos require splicing of zygotically transcribed pre-mRNAs, 36 these combined data lead to the interpretation that coilin supports the high embryonic demand for splicing by promoting rapid snRNP assembly in CBs. This explanation could easily also apply to mouse embryos.…”

“…6,7,17,19 The domain critical for CB formation is the N-terminal 92 amino acids, which is necessary for coilin self-interaction, coilin targeting to CBs, and de novo CB formation. [10][11][12] However, the ability of the N-terminus to form CBs is modulated by the C-terminus and even subtle differences in C-terminal sequence between mouse and human coilin affect CB nucleation.…”

Coordinating cell cycle-regulated histone gene expression through assembly and function of the Histone Locus Body

“…96 Indeed, recent data demonstrate that Cajal bodies enhance the assembly of mature snRNPs both in mammalian cells and early zebrafish embryos. 97,98 In this section we will summarize recent experiments that address how the HLB contributes to histone mRNA biosynthesis, with an emphasis on the role of FLASH and U7 snRNP in the pre-mRNA processing reaction.…”