Identification of human microRNA targets from isolated argonaute protein complexes.
MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that regulate gene expression on the level of translation and/or mRNA stability. Mammalian miRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences in the 3' untranslated region (UTR) of specific target mRNAs. Computer algorithms based on factors such as free binding energy or sequence conservation have been used to predict miRNA target mRNAs. Based on such predictions, up to one third of all mammalian mRNAs seem to be under miRNA regulation. However, due to the low degree of complementarity between the miRNA and its target, such computer programs are often imprecise and therefore not very reliable. Here we report the first biochemical identification approach of miRNA targets from human cells. Using highly specific monoclonal antibodies against members of the Ago protein family, we co-immunoprecipitate Ago-bound mRNAs and identify them by cloning. Interestingly, most of the identified targets are also predicted by different computer programs. Moreover, we randomly analyzed six different target candidates and were able to experimentally validate five as miRNA targets. Our data clearly indicate that miRNA targets can be experimentally identified from Ago complexes and therefore provide a new tool to directly analyze miRNA function.
Beitzinger
Michaela
M
Laboratory of RNA Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Peters
Lasse
L
Zhu
Jia Yun
JY
Kremmer
Elisabeth
E
Meister
Gunter
G
D000818:Animals; D060565:Argonaute Proteins; D002460:Cell Line; D015852:Eukaryotic Initiation Factor-2 / Q000302:isolation & purification / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats
C115848:AGO1 protein, human; C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs; D012333:RNA, Messenger