m₃G Cap Hypermethylation of U1 Small Nuclear Ribonucleoprotein (snRNP) In Vitro: Evidence that the U1 Small Nuclear RNA-(Guanosine-N2)-Methyltransferase Is a Non-snRNP Cytoplasmic Protein That Requires a Binding Site on the Sm Core Domain

Abstract

The RNA components of small nuclear ribonucleoproteins (U snRNPs) possess a characteristic 5'-terminal trimethylguanosine cap structure (m3G cap). This cap is an important component of the nuclear localization signal of U snRNPs. It arises by hypermethylation of a cotranscriptionally added m7G cap. Here we describe an in vitro assay for the hypermethylation, which employs U snRNP particles reconstituted in vitro from purified components and subsequent analysis by m3G cap-specific immunoprecipitation. Complementation studies in vitro revealed that both cytosol and S-adenosylmethionine are required for the hypermethylation of an m7G-capped U1 snRNP reconstituted in vitro, indicating that the U1 snRNA-(guanosine-N2)-methyltransferase is a trans-active non-snRNP protein. Chemical modification revealed one cytoplasmic component required for hypermethylation and one located on the snRNP: these components have different patterns of sensitivity to modification by N-ethylmaleimide and iodoacetic acid (IAA). In the presence of cytosol and S-adenosylmethionine, an intact Sm core domain is a necessary and sufficient substrate for cap hypermethylation. These data, together with our observation that isolated native U1 snRNPs but not naked U1 RNA inhibit the trimethylation of in vitro-reconstituted U1 snRNP, indicate that the Sm core binds the methyltransferase specifically. Moreover, isolated native U2 snRNP also inhibits trimethylation of U1 snRNP, suggesting that other Sm-class U snRNPs might share the same methyltransferase. IAA modification of m7G-capped U1 snRNPs inhibited hypermethylation when they were microinjected into Xenopus oocytes and consequently also inhibited nuclear import. In contrast, modification with IAA of m3G-capped U1 snRNPs reconstituted in vitro did not interfere with their nuclear transport in oocytes. These data suggest that m3G cap formation and nuclear transport of U1 snRNPs are mediated by distinct factors, which require distinct binding sites on the Sm core of U1 snRNP.

Mammalian and yeast U3 snoRNPs are matured in specific and related nuclear compartments

“…While pre‐immune serum did not reveal any signal, both immune sera revealed a similar pattern. As expected from its predicted role in snRNA cap modification (Mattaj, 1986; Plessel et al ., 1994), some signal was detected in the cytoplasm and this will be described in more detail elsewhere. In addition, the two antibodies labeled several bright spots in the nucleus, and double labeling with fibrillarin antibodies demonstrated that these were Cajal bodies (Figure 5B).…”

“…Other RNAs like tRNAs or SRP RNA are also thought to be matured in the nucleolus (Bertrand et al, 1998;Politz et al, 2000;Grosshans et al, 2001). Interestingly, in Archaea, some RNA precursors generate both snoRNA and either rRNA or tRNA (Ome et al, 2000;d'Orval et al, 2001;Tang et al, 2002).…”

A Sm-like protein complex that participates in mRNA degradation

“…These proteins play a role in the biogenesis of the corresponding snRNPs. They associate with the U snRNAs in the cytoplasm, trigger snRNA cap hypermethylation and are part of the snRNP nuclear import signal (Mattaj et al ., 1993a; Plessel et al ., 1994). Yeast Sm proteins are also required for the stable accumulation of the associated U snRNAs in vivo (Rymond, 1993; Roy et al ., 1995; Bordonne and Tarassov, 1996).…”

Mutations in the non-codingRNU4ATACgene affect the homeostasis and function of the Integrator complex

“…The association of the common Sm core proteins with the Sm site of the snRNA occurs in the cytoplasm and is regulated by the survival motor neuron (SMN) complex and protein arginine methyltransferases (14)(15)(16)(17). The binding of the Sm core to the snRNA is required for the hypermethylation of the m 7 G cap structure to an m 3 G cap by the Tgs1 (trimethylguanosine synthase) hypermethylase (18)(19)(20) and for the 3 end trimming of the extra nucleotides by the TOE1 deadenylase (21,22).…”