Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes.
In Xenopus oocytes in vitro transcribed mouse U7 RNA is assembled into small nuclear ribonucleoproteins (snRNPs) that are functional in histone RNA 3' processing. If the special Sm binding site of U7 (AAUUUGUCUAG, U7 Sm WT) is converted into the canonical Sm sequence derived from the major snRNAs (AAUUUUUGGAG, U7 Sm OPT) the RNA assembles into a particle which accumulates more efficiently in the nucleus, but which is non-functional. U7 RNA with a heavily mutated Sm binding site (AACGCGUCAUG, U7 Sm MUT) is deficient in nuclear accumulation and function. By UV cross-linking U7 Sm WT RNA can be linked to three proteins, i.e. the common snRNP proteins G and B/B' and an apparently U7-specific protein of 40 kDa. As a result of altering the Sm binding site, U7 Sm OPT RNA cannot be cross-linked to the 40 kDa protein and no cross-links are obtained with U7 Sm MUT RNA. The fact that the Sm site also interacts with at least one U7-specific protein is so far unique to U7 RNA and may provide an explanation for the atypical sequence of this site. All described RNA-protein interactions, including that with the 40 kDa protein, already occur in the cytoplasm. An additional cytoplasmic photoadduct obtained with U7 Sm WT and U7 Sm OPT, but not U7 Sm MUT, RNAs is indicative of a protein of 60-80 kDa. The m7G cap structure of U7 Sm WT and U7 Sm OPT RNA becomes hypermethylated. However, the 3mG cap enhances, but is not required for, nuclear accumulation. Finally, U7 Sm WT RNA is functional in histone RNA processing even when bearing an ApppG cap.
Stefanovic
B
B
Abteilung für Entwicklungsbiologie, Universität Bern, Switzerland.
Hackl
W
W
Lührmann
R
R
Schümperli
D
D
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D002467:Cell Nucleus / Q000378:metabolism; D003432:Cross-Linking Reagents; D005260:Female; D006657:Histones / Q000235:genetics; D066298:In Vitro Techniques; D008745:Methylation; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012342:RNA, Small Nuclear / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D017381:Spliceosomes / Q000378:metabolism; D014981:Xenopus
D003432:Cross-Linking Reagents; D006657:Histones; D012315:RNA Caps; D012342:RNA, Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear