TY - JOUR
T1 - Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3′ processing in vitro as ribonucleoprotein particles
AU - Wieben, Eric D.
AU - Nenninger, Joan M.
AU - Pederson, Thoru
N1 - Funding Information:
This investigation was supported by American Cancer Society grant CD-126 to T.P. E.D.W. was the recipient of a postdoctoral fellowship from NIH (CA-06751). A brief account of this work was presented at the American Societ’y for Cell Biology meeting, Kansas City. November 12-16. 1984, and has been published in abstract form (Kunkel & Pederson. 1984). We gratefully acknowledge the participation of Steven Madore in the initial phase of this work, and the essential advice and help of Gary Kunkel in construction of the Ml3-U2 DNA clone. We are especially grateful to Reinhard Luhrmann for his generous gift of trimethylguanosine cap antibody and lllf Pettersson, Lennart Philipson and Gunnar West,in for kindly providing the human U2 DNA clone.
PY - 1985/5/5
Y1 - 1985/5/5
N2 - Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 °C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3′ processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5′ trimethylguanosine caps would be expected to compete for a 5′ processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.
AB - Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 °C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3′ processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5′ trimethylguanosine caps would be expected to compete for a 5′ processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.
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U2 - 10.1016/0022-2836(85)90281-5
DO - 10.1016/0022-2836(85)90281-5
M3 - Article
C2 - 2409291
AN - SCOPUS:0022401689
SN - 0022-2836
VL - 183
SP - 69
EP - 78
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -