Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm

Abstract

Biogenesis of functional spliceosomal small nuclear RNAs (snRNAs) includes the post-transcriptional covalent modi®cation of numerous internal nucleotides. We have recently demonstrated that synthesis of 2¢-O-methylated nucleotides and pseudouridines in the RNA polymerase II-synthesized Sm snRNAs is directed by sequence-speci®c guide RNAs. Here, we provide evidence supporting the notion that modi®ca-tion of Sm snRNAs occurs in nucleoplasmic Cajal bodies (CBs), where modi®cation guide RNAs accumulate. We show that short fragments of Sm snRNAs are correctly and ef®ciently modi®ed when targeted to CBs, but not when these same fragments are targeted to the nucleolus. We also demonstrate that internal modi®cation of the U2 snRNA occurs exclusively after nuclear import of the newly assembled Sm snRNP from the cytoplasm. Finally, we show that p80 coilin, the CB marker protein, is not required for snRNA modi®cation. In coilin knockout cells, Sm snRNAs and their modi®cation guide RNAs colocalize in residual CBs, which do not stockpile ®brillarin and fail to recruit the U3 small nucleolar RNA.

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

“…Moreover, recent work has implicated the CB as a site of snRNA modification (Jady et al 2003) and assembly of snRNP-specific proteins (Nesic et al 2004;Tanackovic and Kramer 2005). These results raise several mechanistic questions.…”

“…Upon nuclear reentry, newly assembled snRNPs target first to the CBs (Sleeman and Lamond 1999), where the snRNAs are likely modified (Jady et al 2003) and assembled into mature RNPs (Nesic et al 2004;Tanackovic and Kramer 2005). These 2′-O-methyl sugar-and pseudouridine-base modifications of the U snRNPs (Darzacq et al 2002;Jady et al 2003;Verheggen et al 2002) are guided by small CB-specific RNAs (scaRNAs) and are crucial for proper pre-mRNA splicing in vivo (Yu et al 1998).…”

“…These 2′-O-methyl sugar-and pseudouridine-base modifications of the U snRNPs (Darzacq et al 2002;Jady et al 2003;Verheggen et al 2002) are guided by small CB-specific RNAs (scaRNAs) and are crucial for proper pre-mRNA splicing in vivo (Yu et al 1998). …”

“…Coilin is highly conserved among vertebrates and clear homologs can be found in plants (Tucker and Matera 2004), but the gene has not been identified in worms and flies. Although coilin appears to be absent from the genomes of worms and flies, structures similar to CBs have been identified in various insects including Drosophila (Gall et al 1995;Jady et al 2003;Yannoni and White 1997). Moreover, coilin is required for the accumulation of U snRNPs within the CBs of amphibians and mammals (Bauer and Gall 1997;Hebert et al 2001;Tucker et al 2001).…”

Targeting SMN to Cajal bodies and nuclear gems during neuritogenesis

“…In order to meet this new demand, factors involved in pre-mRNA processing must also be synthesized. Cajal bodies (CBs) are nuclear structures involved in the biogenesis of RNA-processing factors (Verheggen et al 2002;Jady et al 2003) and are prominent features of mammalian neurons (Cajal 1910;Lafarga et al 1983;Peters et al 1991;Pena et al 2001). While little is known about nuclear remodeling events during neuronal differentiation, late-stage neurons are characterized by larger cell bodies with smaller and diffusely organized splicing factor compartments (Pena et al 2001).…”