Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells.
A 21-base pair RNA duplex that perfectly matches an endogenous target mRNA selectively degrades the mRNA and suppresses gene expression in mammalian tissue culture cells. A single base mismatch with the target is believed to protect the mRNA from degradation, making this type of interference highly specific to the targeted gene. A short RNA with mismatches to a target sequence present in multiple copies in the 3'-untranslated region of an exogenously expressed gene can, however, silence it by translational repression. Here we report that a mismatched RNA, targeted to a single site in the coding sequence of an endogenous gene, can efficiently silence gene expression by repressing translation. The antisense strand of such a mismatched RNA requires a 5'-phosphate but not a 3'-hydroxyl group. G.U wobble base pairing is tolerated as a match for both RNA degradation and translation repression. Together, these findings suggest that a small inhibitory RNA duplex can suppress expression of off-target cellular proteins by RNA degradation or translation repression. Proper design of experimental small inhibitory RNAs or a search for targets of endogenous micro-RNAs must therefore take into account that these short RNAs can affect expression of cellular genes with as many as 3-4 base mismatches and additional G.U mismatches.
Saxena
Sandeep
S
Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908, USA.
Jónsson
Zophonías O
ZO
Dutta
Anindya
A
D020413:3' Untranslated Regions / Q000235:genetics; D020137:Base Pair Mismatch; D020029:Base Pairing; D001483:Base Sequence; D002460:Cell Line; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D010710:Phosphates / Q000737:chemistry; D014176:Protein Biosynthesis / Q000187:drug effects*; D016372:RNA, Antisense / Q000494:pharmacology; D012333:RNA, Messenger / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000494:pharmacology*; D034743:RNA-Induced Silencing Complex / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection
D020413:3' Untranslated Regions; D010710:Phosphates; D016372:RNA, Antisense; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex