Identification, cloning, and functional analysis of the human U6 snRNA-specific terminal uridylyl transferase.
Mammalian cells contain a highly specific terminal uridylyl transferase (TUTase) that exclusively accepts U6 snRNA as substrate. This enzyme, termed U6-TUTase, was purified from HeLa cell extracts and analyzed by microsequencing. All sequenced peptides matched a unique human cDNA coding for a previously unknown protein. Domain structure analysis revealed that the U6-TUTase also belongs to the well-characterized poly(A) polymerase protein superfamily. However, by amino acid sequence as well as RNA-binding motifs, human U6-TUTase is highly divergent from both the poly(A) polymerases and from the TUTases identified within the editing complexes of trypanosomes. After cloning, the recombinant U6-TUTase was expressed in HeLa cells. Analysis of its catalytical activity confirmed the identity of the cloned protein as U6-TUTase, exhibiting the same exclusive substrate specificity for U6 snRNA as the endogenous enzyme. That unique selectivity even excluded as substrate U6atac RNA, the functional homolog of the minor spliceosome. Finally, RNAi knockdown experiments revealed that U6-TUTase is essential for cell proliferation. Surprisingly, large amounts of the recombinant enzyme were found to accumulate within nucleoli.
Trippe
Ralf
R
Lehrstuhl für Biochemie I, Ruhr-Universität, Bochum, Germany.
Guschina
Elena
E
Hossbach
Markus
M
Urlaub
Henning
H
Lührmann
Reinhard
R
Benecke
Bernd-Joachim
BJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002466:Cell Nucleolus / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry; D034622:RNA Interference; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013347:Subcellular Fractions / Q000378:metabolism; D016335:Zinc Fingers
D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; C405486:U6 small nuclear RNA; D006639:Histidine; D012316:RNA Nucleotidyltransferases