ACCURATE MODIFICATION OF THE TRYPANOSOME SPLICED LEADER CAP STRUCTURE IN A HOMOLOGOUS CELL-FREE SYSTEM

被引:26
作者
ULLU, E
TSCHUDI, C
机构
[1] YALE UNIV,SCH MED,DEPT INTERNAL MED,NEW HAVEN,CT 06520
[2] YALE UNIV,SCH MED,DEPT CELL BIOL,NEW HAVEN,CT 06520
关键词
D O I
10.1074/jbc.270.35.20365
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
During RNA maturation in trypanosomatid protozoa, trans-splicing transfers the spliced leader (SL) sequence and its cap from the SL RNA to the 5' end of all mRNAs. In Trypanosoma brucei and Crithidia fasciculata the SL RNA has an unusual cap structure with four methylated nucleotides following the 7-methylguanosine residue (cap 4). Since modification of the 5' end of the SL RNA is a pre-requisite for trans-splicing activity in T. brucei, we have begun to characterize the enzyme(s) involved in this process. Here we report the development of a T. brucei cell-free system for modification of the cap of the SL RNA. Analysis of the nucleotide composition of the in vitro generated cap structure by two-dimensional thin layer chromatography established that the in vitro reaction is accurate. Cap 4 formation requires the SL RNA to be in a ribonucleoprotein particle and can be inhibited by annealing a complementary 2'-O-methyl RNA oligonucleotide to nucleotides 7-18 of the SL RNA. Methylation of the 5' end of the SL RNA is also required for trans-splicing in T. cruzi and Leishmania amazonensis and cell-free extracts from C. fasciculata and L. amazonensis are capable of modifying the cap structure on the T. brucei SL ribonucleoprotein particle.
引用
收藏
页码:20365 / 20369
页数:5
相关论文
共 26 条
[11]  
HARRIS KA, 1995, RNA, V1, P351
[12]   REQUIREMENT OF A POLYPYRIMIDINE TRACT FOR TRANSSPLICING IN TRYPANOSOMES - DISCRIMINATING THE PARP PROMOTER FROM THE IMMEDIATELY ADJACENT 3' SPLICE ACCEPTOR SITE [J].
HUANG, J ;
VANDERPLOEG, LHT .
EMBO JOURNAL, 1991, 10 (12) :3877-3885
[13]   A NUCLEAR CAP-BINDING PROTEIN COMPLEX INVOLVED IN PRE-MESSENGER-RNA SPLICING [J].
IZAURRALDE, E ;
LEWIS, J ;
MCGUIGAN, C ;
JANKOWSKA, M ;
DARZYNKIEWICZ, E ;
MATTAJ, IW .
CELL, 1994, 78 (04) :657-668
[14]   INVIVO LABELING OF INTERMEDIATES IN THE DISCONTINUOUS SYNTHESIS OF MESSENGER-RNAS IN TRYPANOSOMA-BRUCEI [J].
LAIRD, PW ;
ZOMERDIJK, JCBM ;
DEKORTE, D ;
BORST, P .
EMBO JOURNAL, 1987, 6 (04) :1055-1062
[15]   TRYPANOSOMA-BRUCEI SPLICED-LEADER RNA METHYLATIONS ARE REQUIRED FOR TRANSSPLICING INVIVO [J].
MCNALLY, KP ;
AGABIAN, N .
MOLECULAR AND CELLULAR BIOLOGY, 1992, 12 (11) :4844-4851
[16]   PSEUDOURIDINE MODIFICATION OF U5-RNA IN RIBONUCLEOPROTEIN-PARTICLES ASSEMBLED INVITRO [J].
PATTON, JR .
MOLECULAR AND CELLULAR BIOLOGY, 1991, 11 (12) :5998-6006
[17]   TRYPANOSOME MESSENGER-RNAS HAVE UNUSUAL CAP-4 STRUCTURES ACQUIRED BY ADDITION OF A SPLICED LEADER [J].
PERRY, KL ;
WATKINS, KP ;
AGABIAN, N .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1987, 84 (23) :8190-8194
[18]   M(3)G CAP HYPERMETHYLATION OF U1 SMALL NUCLEAR RIBONUCLEOPROTEIN (SNRNP) IN-VITRO - EVIDENCE THAT THE U1 SMALL NUCLEAR RNA-(GUANOSINE-N2)-METHYLTRANSFERASE IS A NON-SNRNP CYTOPLASMIC PROTEIN THAT REQUIRES A BINDING-SITE ON THE SM CORE DOMAIN [J].
PLESSEL, G ;
FISCHER, U ;
LUHRMANN, R .
MOLECULAR AND CELLULAR BIOLOGY, 1994, 14 (06) :4160-4172
[19]  
Reddy R, 1988, STRUCTURE FUNCTION M, P1, DOI 10.1007/978-3-642-73020-7_1
[20]   MESSENGER-RNA CAP BINDING-PROTEINS - ESSENTIAL FACTORS FOR INITIATING TRANSLATION [J].
SHATKIN, AJ .
CELL, 1985, 40 (02) :223-224