The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.
Mitochondrial mRNA editing in Trypanosoma brucei requires the specific interaction of a guide RNA with its cognate mRNA. Hundreds of gRNAs are involved in the editing process, each needing to target their specific editing domain within the target message. We hypothesized that the structure surrounding the mRNA target may be a limiting factor and involved in the regulation process. In this study, we selected four mRNAs with distinct target structures and investigated how sequence and structure affected efficient gRNA targeting. Two of the mRNAs, including the ATPase subunit 6 and ND7-550 (5' end of NADH dehydrogenase subunit 7) that have open, accessible anchor binding sites show very efficient gRNA targeting. Electrophoretic mobility shift assays indicate that the cognate gRNA for ND7-550 had 10-fold higher affinity for its mRNA than the A6 pair. Surface plasmon resonance studies indicate that the difference in affinity was due to a four-fold faster association rate. As expected, mRNAs with considerable structure surrounding the anchor binding sites were less accessible and had very low affinity for their cognate gRNAs. In vitro editing assays indicate that efficient pairing is crucial for gRNA directed cleavage. However, only the A6 substrate showed gRNA-directed cleavage at the correct editing site. This suggests that different gRNA/mRNA pairs may require different "sets" of accessory factors for efficient editing. By characterizing a number of different gRNA/mRNA interactions, we may be able to define a "bank" of RNA editing substrates with different putative chaperone and other co-factor requirements. This will allow the more efficient identification and characterization of transcript specific RNA editing accessory proteins.
Reifur
Larissa
L
Comparative Medicine and Integrative Biology Program, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, United States of America.
Yu
Laura E
LE
Cruz-Reyes
Jorge
J
Vanhartesvelt
Michelle
M
Koslowsky
Donna J
DJ
D001483:Base Sequence; D001665:Binding Sites; D007700:Kinetics; D016830:Kinetoplastida; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017393:RNA Editing; D017394:RNA, Guide, Kinetoplastida / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D017394:RNA, Guide, Kinetoplastida
Cell. 1990 Jun 15;61(6):1001-11
1693545
Methods. 2002 Feb;26(2):95-104
12054886
Mol Cell Biol. 1997 Sep;17(9):5377-85
9271415
Cell. 1990 Sep 7;62(5):901-11
2393904
Nat Biotechnol. 1998 Jan;16(1):64-8
9447596
Annu Rev Microbiol. 1994;48:713-42
7826024
Nucleic Acids Res. 2001 Jun 15;29(12):2575-80
11410666
J Mol Biol. 2001 Jul 27;310(5):1125-34
11502000
J Biol Chem. 2005 Jan 28;280(4):2429-38
15504736
Curr Opin Biotechnol. 1997 Feb 1;8(1):50-7
9013659
Cell. 2006 Aug 25;126(4):701-11
16923390
J Mol Biol. 2006 Jun 9;359(3):585-96
16631792
Science. 1992 May 15;256(5059):992-7
1589782
Nucleic Acids Res. 2002 Jan 15;30(2):447-55
11788706
EMBO J. 2001 Mar 15;20(6):1394-404
11250905
Int J Parasitol. 2001 Nov;31(13):1403-15
11595226
Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W577-81
15980540
PLoS One. 2008 Feb 13;3(2):e1566
18270563
RNA. 2001 Dec;7(12):1803-16
11780636
Mol Cell Biol. 2002 Jul;22(13):4652-60
12052873
Nucleic Acids Res. 1999 Feb 1;27(3):778-87
9889273
Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9218-22
7524072
RNA. 2003 Apr;9(4):457-68
12649497
RNA. 1997 Apr;3(4):429-37
9085849
EMBO J. 1997 Jul 1;16(13):4069-81
9233816
RNA. 2003 Jan;9(1):62-76
12554877
Mol Cell Biol. 1988 Mar;8(3):1259-65
2452974
RNA. 2003 Apr;9(4):469-83
12649498
Trends Plant Sci. 2008 Jul;13(7):375-82
18550416
Science. 1996 Aug 30;273(5279):1189-95
8703045
Cell. 1991 Nov 1;67(3):537-46
1718605
Mol Cell Biol. 1992 May;12(5):2043-9
1373804
Mol Cell. 1998 Feb;1(3):401-9
9660924
Mol Cell Biol. 2008 Jan;28(1):122-30
17954557
Cell. 1990 Jan 26;60(2):189-98
1688737
Methods. 2003 Feb;29(2):167-74
12606222
EMBO Rep. 2006 Nov;7(11):1128-33
17008930
RNA. 2008 Oct;14(10):2195-211
18772247
EMBO J. 1992 Dec;11(12):4429-38
1330537
J Mol Biol. 2008 Aug 1;381(1):35-48
18572190
Cell. 1987 May 8;49(3):337-45
3568129
RNA. 2008 Jun;14(6):1069-80
18441045
Annu Rev Cell Dev Biol. 2007;23:175-205
17506695
J Biol Chem. 2007 Feb 16;282(7):4265-4276
17158098
RNA Biol. 2004 May;1(1):28-34
17194935
Trends Biochem Sci. 2005 Feb;30(2):97-105
15691655
Nucleic Acids Res. 2000 Nov 1;28(21):4113-24
11058107
Mol Cell Biol. 2002 Mar;22(5):1567-76
11839822
Cell. 2008 Sep 19;134(6):899-902
18805079