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Functional characterisation of microRNA-containing Argonaute protein complexes
Functional characterisation of microRNA-containing Argonaute protein complexes
microRNAs (miRNAs) are small non-coding RNAs of 21-24 nt in size, which are endogenously expressed in higher eukaryotes and play important roles in processes such as tissue development and stress response and in several diseases including cancers. In mammals, miRNAs guide proteins of the Argonaute family (Ago proteins) to partially complementary sequences typically located in the 3’-untranslated regions (3’-UTRs) of specific target mRNAs, leading to translational repression or mRNA degradation. To gain further insight into the function of human miRNAs, we analyzed the protein as well as the RNA composition of miRNA-Ago protein complexes in molecular detail. To identify novel Ago-interacting proteins, we isolated Ago complexes and investigated them by mass spectrometry and co-immunoprecipitation experiments. We found that trinucleotide repeat-containing 6B (TNRC6B), Moloney leukemia virus 10 (MOV10), RNA binding motif protein 4 (RBM4) and Importin 8 (Imp8) interact with human Ago proteins. Moreover, using RNA interference and EGFP and dual luciferase reporter assays, we demonstrated that these factors are required for miRNA function, indicating that we have identified new components of the miRNA pathway. Intriguingly, depletion of Imp8 does not affect the levels of mature miRNAs or the interaction of miRNAs with Ago proteins, but is required for efficient association of Ago-miRNA complexes with their target mRNAs. Thus, Imp8 is the first factor acting at the level of target mRNA binding, establishing a novel layer of regulation for the miRNA pathway. Imp8 is an Importin-β-like protein, which has previously been implicated in nuclear import of substrate proteins. In line with these results, we demonstrated that a detectable fraction of Ago2 localizes to the nucleus of human cells. Moreover, knockdown of Imp8 by RNAi reduces the nuclear signal of Ago2, suggesting that Imp8 affects the nuclear localization of Ago2. Therefore, our data suggest that Imp8 has a dual function both in the cytoplasmic miRNA pathway and in nuclear transport of Ago proteins. To identify small RNAs, which associate with human Ago proteins, we isolated, cloned and sequenced small RNAs bound to Ago1 and Ago2 complexes. In addition to known miRNAs, we found several small RNAs, which derive from small nucleolar RNAs (snoRNAs). We therefore investigated the function of one particular small RNA, which is derived from the snoRNA ACA45 and showed that it functions like a miRNA. Interestingly, this small RNA is processed by the miRNA maturation factor Dicer, but does not require the microprocessor complex that is essential for processing of primary miRNA transcripts. Thus, we have identified a novel biogenesis pathway of a new class of small RNAs that can function like miRNAs. To experimentally identify mRNAs that are stably associated with miRNA-Ago protein complexes, we isolated and analyzed Ago1 and Ago2-bound mRNAs by cloning and sequencing and by microarray hybridization techniques. Using dual luciferase reporter assays, we demonstrated that many Ago-associated mRNAs are indeed miRNA targets. Therefore, we have developed a method allowing for the identification of miRNA target mRNAs from cell lines or tissues of interest independently of computational predictions. In a project that was independent of our studies on Ago protein complexes, we investigated structural and functional requirements for the activity of small interfering RNAs (siRNAs). siRNAs are small double-stranded RNAs of appr. 21 nt in size, which trigger the sequence-specific endonucleolytic degradation of perfectly complementary target transcripts upon binding to Ago2. However, both single strands of a siRNA duplex can potentially have unwanted “off-target effects” by repressing partially complementary target mRNAs through binding to their 3’-UTRs. We therefore developed a method to selectively inhibit the activity of the siRNA strand that is dispensable for target silencing (“passenger strand”) through chemical modification of its 5’-end. This method could be a useful tool for the design of highly specific siRNAs. Taken together, we have analyzed the composition of Ago-miRNA protein complexes by a variety of methods and identified novel protein factors of the miRNA pathway, a novel class of small RNAs as well as a panel of previously unknown miRNA target mRNAs. The techniques for the purification and the analysis of Ago complexes that were developed in this study will provide useful tools for future analyses of miRNA pathway factors, small RNAs and miRNA target mRNAs from any tissue or cell line of interest.
microRNA, miRNA, RNAi, Argonaute
Weinmann, Lasse
2009
Englisch
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Weinmann, Lasse (2009): Functional characterisation of microRNA-containing Argonaute protein complexes. Dissertation, LMU München: Fakultät für Biologie
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Abstract

microRNAs (miRNAs) are small non-coding RNAs of 21-24 nt in size, which are endogenously expressed in higher eukaryotes and play important roles in processes such as tissue development and stress response and in several diseases including cancers. In mammals, miRNAs guide proteins of the Argonaute family (Ago proteins) to partially complementary sequences typically located in the 3’-untranslated regions (3’-UTRs) of specific target mRNAs, leading to translational repression or mRNA degradation. To gain further insight into the function of human miRNAs, we analyzed the protein as well as the RNA composition of miRNA-Ago protein complexes in molecular detail. To identify novel Ago-interacting proteins, we isolated Ago complexes and investigated them by mass spectrometry and co-immunoprecipitation experiments. We found that trinucleotide repeat-containing 6B (TNRC6B), Moloney leukemia virus 10 (MOV10), RNA binding motif protein 4 (RBM4) and Importin 8 (Imp8) interact with human Ago proteins. Moreover, using RNA interference and EGFP and dual luciferase reporter assays, we demonstrated that these factors are required for miRNA function, indicating that we have identified new components of the miRNA pathway. Intriguingly, depletion of Imp8 does not affect the levels of mature miRNAs or the interaction of miRNAs with Ago proteins, but is required for efficient association of Ago-miRNA complexes with their target mRNAs. Thus, Imp8 is the first factor acting at the level of target mRNA binding, establishing a novel layer of regulation for the miRNA pathway. Imp8 is an Importin-β-like protein, which has previously been implicated in nuclear import of substrate proteins. In line with these results, we demonstrated that a detectable fraction of Ago2 localizes to the nucleus of human cells. Moreover, knockdown of Imp8 by RNAi reduces the nuclear signal of Ago2, suggesting that Imp8 affects the nuclear localization of Ago2. Therefore, our data suggest that Imp8 has a dual function both in the cytoplasmic miRNA pathway and in nuclear transport of Ago proteins. To identify small RNAs, which associate with human Ago proteins, we isolated, cloned and sequenced small RNAs bound to Ago1 and Ago2 complexes. In addition to known miRNAs, we found several small RNAs, which derive from small nucleolar RNAs (snoRNAs). We therefore investigated the function of one particular small RNA, which is derived from the snoRNA ACA45 and showed that it functions like a miRNA. Interestingly, this small RNA is processed by the miRNA maturation factor Dicer, but does not require the microprocessor complex that is essential for processing of primary miRNA transcripts. Thus, we have identified a novel biogenesis pathway of a new class of small RNAs that can function like miRNAs. To experimentally identify mRNAs that are stably associated with miRNA-Ago protein complexes, we isolated and analyzed Ago1 and Ago2-bound mRNAs by cloning and sequencing and by microarray hybridization techniques. Using dual luciferase reporter assays, we demonstrated that many Ago-associated mRNAs are indeed miRNA targets. Therefore, we have developed a method allowing for the identification of miRNA target mRNAs from cell lines or tissues of interest independently of computational predictions. In a project that was independent of our studies on Ago protein complexes, we investigated structural and functional requirements for the activity of small interfering RNAs (siRNAs). siRNAs are small double-stranded RNAs of appr. 21 nt in size, which trigger the sequence-specific endonucleolytic degradation of perfectly complementary target transcripts upon binding to Ago2. However, both single strands of a siRNA duplex can potentially have unwanted “off-target effects” by repressing partially complementary target mRNAs through binding to their 3’-UTRs. We therefore developed a method to selectively inhibit the activity of the siRNA strand that is dispensable for target silencing (“passenger strand”) through chemical modification of its 5’-end. This method could be a useful tool for the design of highly specific siRNAs. Taken together, we have analyzed the composition of Ago-miRNA protein complexes by a variety of methods and identified novel protein factors of the miRNA pathway, a novel class of small RNAs as well as a panel of previously unknown miRNA target mRNAs. The techniques for the purification and the analysis of Ago complexes that were developed in this study will provide useful tools for future analyses of miRNA pathway factors, small RNAs and miRNA target mRNAs from any tissue or cell line of interest.