Library

Notes: Summary U7-snRNA is an essential component of the RNA processing machinery which generates the 3′ end of mature histone mRNA in the sea urchin. U7-snRNP is classified as a member of the Sm-type U-snRNP family by virtue of its recognition by both anti-thrimethylguanosine and anti-Sm antibodies. We have analyzed the function/structure relationship of the U7-snRNP by mutagenesis experiments. These suggest that the U7-snRNP particle of the sea urchin has three important domains. 1. The 5′ terminal sequences, nucleotides 1–9, are accessible to micronuclease while the remainder of the RNA is highly protected and hence presumably bound up with proteins. The complementarities of the U7-RNA 5′ terminal sequences to the histone pre-mRNA are of functional importance. 2. Nucleotides 9–20 contain a sequence mediating Sm-protein binding. The complementarities between the U7-RNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a minor, if any, role in 3′ processing. 3. The terminal palindrome of U7-RNA must be maintained as structure in order for the U7-RNP to work, but its sequence can be drastically altered without any observable effect on snRNP assembly or in 3′ processing. In addition to the Sm-type snRNPs, at least one more factor is involved in 3′ processing of histone pre-mRNAs. The corresponding factor from HELA cell nuclear extracts can be completely inactivated by mild heat treatment, but is resistant to digestion with micrococconuclease. It cannot be immunoprecipitated by antisera of the Sm-serotype. Extracts have been treated with micrococconuclease or depleted with anti-Sm antisera and can be complemented with a heat treated extract, whereas micrococconuclease treated and Sm-antibody treated extracts cannot complement each other. Both snRNP (the presumed human homologue of the U7-snRNP of the sea urchin) and the heat-labile factor show closely similar properties when fractionated on DEAE, heparin and mono Q columns. Fractions, some 5000 x purified still contain both heat-labile factor and snRNP activity. When analyzed by molecular sieving, the heat-labile component distributes bimodally, the smaller molecule possessing an apparant molecular weight of approximately 40 kD, the larger component fractionating as a 3–4×105 kD particle, indistinguishable in this property from the putative U7-snRNP, both components requiring complementation with snRNPs to show activity in 3′ processing. The activity (or concentration) of the heat-labile component is the regulatory element of the 3′ processing reaction. It is strongly down-regulated in a mutant mouse mastocytoma cell line which is prevented from proliferation by a temperature shift (Lüscher B. and Schümperli D., EMBO Journal 6, 1721–1726 (1986) but not in cells in which DNA synthesis is abolished with inhibitors.