FACILITATION OF HAMMERHEAD RIBOZYME CATALYSIS BY THE NUCLEOCAPSID PROTEIN OF HIV-1 AND THE HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN A1

被引:139
作者
BERTRAND, EL [1 ]
ROSSI, JJ [1 ]
机构
[1] BECKMAN RES INST CITY HOPE,DEPT MOLEC GENET,DUARTE,CA 91010
关键词
HAMMERHEAD RIBOZYME; HNRNP; NUCLEOCAPSID PROTEIN;
D O I
10.1002/j.1460-2075.1994.tb06585.x
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
In order to improve the activity of hammerhead ribozymes in vivo, we have analyzed the effect of several prototypical RNA binding proteins on the ribozyme cleavage reaction: bacteriophage T4 gene 32 protein (gp32), hnRNP Al (Al) and the nucleocapsid protein of HIV-1 (NCp7). We show that, while gp32 has no effect on the cleavage reaction, Al and NCp7 affect different steps of the reaction. Moreover, some of these effects depend upon the ribozyme - substrate hybrid length. A1 and NCp7 inhibit the reaction of the least stable ribozyme-substrate complexes, which have 12 bp of duplex. NCp7, but not Al, inhibits the cleavage of substrates that have long ribozyme-substrate duplexes (17 or 20 bp), while cleavage of complexes having shorter duplexes (13 or 14 bp) is not affected. NCp7 and Al enhance the turnover of ribozymes by increasing the rate of product dissociation, but only when both cleavage products are bound with less than or equal to 7 bp. Al and NCp7 enhance ribozyme binding to long substrates, such as mRNAs, the structure of which otherwise limits ribozyme binding. Therefore, the effects of Al or NCp7 on the different steps of the cleavage reaction define a length of the ribozyme-substrate duplex which allows enhancement of the rate of binding and product release without inhibiting the cleavage step. Interestingly, this duplex length (14 bases, or 7 on each side of the cleavage site) is identical for Al and NCp7. Since Al is thought to interact with most, if not all mRNAs in vivo, it may enhance the intracellular activity of ribozymes targeted against any mRNA. On the other hand, since retroviral nucleocapsid (NC) proteins interact only with the viral genomic RNA in vivo, these results raise the exciting possibility that NCp7 and other NC proteins may specifically enhance the activity of ribozymes targeted against their cognate retroviruses. Overexpression of Al or NCp7, and targeting the ribozyme to a cellular compartment rich in Al or NCp7 may also enhance ribozyme activity in vivo.
引用
收藏
页码:2904 / 2912
页数:9
相关论文
共 64 条
[31]   PRIMARY STRUCTURE AND BINDING-ACTIVITY OF THE HNRNP U-PROTEIN - BINDING RNA THROUGH RGG BOX [J].
KILEDJIAN, M ;
DREYFUSS, G .
EMBO JOURNAL, 1992, 11 (07) :2655-2664
[32]   STUDIES OF THE STRAND-ANNEALING ACTIVITY OF MAMMALIAN HNRNP COMPLEX PROTEIN-A1 [J].
KUMAR, A ;
WILSON, SH .
BIOCHEMISTRY, 1990, 29 (48) :10717-10722
[33]  
LEE CG, 1993, J BIOL CHEM, V268, P13472
[34]   CYTOPLASMIC DELIVERY OF RIBOZYMES LEADS TO EFFICIENT REDUCTION IN ALPHA-LACTALBUMIN MESSENGER-RNA LEVELS IN C127I MOUSE CELLS [J].
LHUILLIER, PJ ;
DAVIS, SR ;
BELLAMY, AR .
EMBO JOURNAL, 1992, 11 (12) :4411-4418
[35]   MODULATION OF EXON SKIPPING AND INCLUSION BY HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN-A1 AND PREMESSENGER RNA SPLICING FACTOR SF2/ASF [J].
MAYEDA, A ;
HELFMAN, DM ;
KRAINER, AR .
MOLECULAR AND CELLULAR BIOLOGY, 1993, 13 (05) :2993-3001
[36]   REGULATION OF ALTERNATIVE PRE-MESSENGER-RNA SPLICING BY HNRNP-A1 AND SPLICING FACTOR-SF2 [J].
MAYEDA, A ;
KRAINER, AR .
CELL, 1992, 68 (02) :365-375
[37]   CROSS-LINKING OF HNRNP PROTEINS TO PRE-MESSENGER-RNA REQUIRES U1 AND U2 SNRNPS [J].
MAYRAND, SH ;
PEDERSON, T .
NUCLEIC ACIDS RESEARCH, 1990, 18 (11) :3307-3318
[38]   HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN-A1 CATALYZES RNA-RNA ANNEALING [J].
MUNROE, SH ;
DONG, XF .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (03) :895-899
[39]   INTERACTIONS OF THE A1 HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN AND ITS PROTEOLYTIC DERIVATIVE, UP1, WITH RNA AND DNA - EVIDENCE FOR MULTIPLE RNA-BINDING DOMAINS AND SALT-DEPENDENT BINDING MODE TRANSITIONS [J].
NADLER, SG ;
MERRILL, BM ;
ROBERTS, WJ ;
KEATING, KM ;
LISBIN, MJ ;
BARNETT, SF ;
WILSON, SH ;
WILLIAMS, KR .
BIOCHEMISTRY, 1991, 30 (11) :2968-2976
[40]   SHUTTLING OF PRE-MESSENGER-RNA BINDING-PROTEINS BETWEEN NUCLEUS AND CYTOPLASM [J].
PINOLROMA, S ;
DREYFUSS, G .
NATURE, 1992, 355 (6362) :730-732