Low-Molecular-Weight Nuclear RNAs (1971)

LOW-MOLECULAR-WEIGHT NUCLEAR RNAs HARRIS BUSCH, TAE SUK ROCHOI, ARCHIE W. PRESTAYKO, HIROTOSHI SHIBATA, STANLEY T. CROOKE, SHUKRI M. EL-KHATIB, YONG C. CHOI, and CHARLES Af. MAURITZEN* The recent development of methods for isolation, purification and high-level labeling of the low-molecular-weight nuclear (LMWN) RNAs with sedimentation coefficients from 4S to 12S has amplified the initial evidence that some of these are specifically localized to the nucleolus and others to the nucleus of mammalian and other cells [1, p. 285; 2-14]. Some RNAs smaller in size than these which contain dihydrouridine or dihydrothymidine have also been reported to be present in the nucleus, specifically in association with the chromatin fraction [15-20]. Although the functions of most of these LMWN RNAs are not defined, their specificity of localization in the nucleolus and the nuclear chromatin fraction along with the number of these molecules (which approximates that of the number of functional genes) has suggested they may have an initiating or structural role in gene readouts or a regulatory role in gene function [8, 10, 2123 ]. The recent findings which have indicated that some low-molecular -weight nucleolar RNAs have specific temporal associations with newly synthesized ribosomal precursors suggest the possibility that they may be involved in specific phases of synthesis or modification of the ribosomal precursors [24]. Number of low-molecular-weight nuclear RNAs.—In a search for a definite role for these molecules in cell function, one of the critical problems has been the clarification of the number of types of these RNA molecules. Although it is not yet possible to specify with precision the total number of low-molecular-weight RNA species in the various fractions of the nucleus, the minimal estimate is eleven (fig. 1), and the maximum is approximately forty-three [1, 25]. None of these values, however, exclude possible microheterogeneity of the various nuclear RNA species. Table 1 indicates that there are at least * Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77025. These studies were supported by the USPHS Cancer Research Center grant CA-10893 and the American Cancer Society grant P-339. Perspectives in Biology and Medicine · Autumn 1971 | 117 Fig. 1.—Polyacrylamide-gel electrophoresis of LMWN RNA. Electrophoresis with these 12 percent gels was carried out for fifty hours with a current of 40 mA at a voltage of 300 ? in a buffer containing 0.02 m Tris, 0.02 m NaCl, and 0.04 m EDTA. eight major groups of the LMWN RNAs up to 8S in size which include transfer RNA molecules in the 4S group and the 5S and 7S(28S) ribosomal RNA. The remaining species of RNA include the 4.5S RNA, 5S RNAn i, Ul and U2 extranucleolar nuclear RNAs and the nucleolar U3 and 8S(28S) RNAs. Although these molecules have been separated into mobility groups by polyacrylamide-gel electrophoresis, additional methods were required for further separation of these RNA groups into individual molecular species [2527 ]. At present, 4.5S RNA1, 4.5S RNA111, UIa and U2 RNA have been obtained in highly purified form (figs. 2-3). Localization of nuclear RNAs.—Analysis of the low-molecularweight RNA of the nucleolus has provided evidence for localization of two types of these RNA fractions, that is, the 8S (28S) RNA and the U3 RNA (fig. 4), regardless of whether nucleoli were isolated by 118 I Harris Busch et al. · Nuclear RNAs TABLE 1»^-Nucleotide Compositions and 3' Terminals of Low-MolecularWeight RNA from Ribosomes, Nucleus, and Nucleolus of Novikoff Hepatoma RNA amp ump GMP CMP AMP +UMP GMP +CMP 3'-Terminal 1.Nuclear 4-8S RNA: 4S ................ Total 4. 5S RNA... 4.5Si ............ 4.5Sn ........... 4.5Sm .......... Total 5S RNA ..... SSi .............. 5Sh ............. 5Sm ............ 7S(28S) ............ Ul ................ U2 ................ U3* ............... 8S(28S)*........... 2.Nucleolar 4-8S RNA 4S* ............... 5S ................ 7SÍ28S) ............ U3* ............... 8S(28S)*........... 3.Ribosomal RNA: 4S ................ 5S ................ 7S(28S) ............ 19.6 23.9 24.8 25.4 26.7 18.8 19.2 20.7 24.0 21.2 20.0 22.0 20.2 18.4 19.5 18.2 19.8 20.2 18.4 15.4 18.2 19.7 20.7 24.6 24.4 26.0...