Analysis of small RNA-Argonaute complexes in different organisms

MicroRNAs (miRNAs) are small, non-coding RNAs of 20-24 nt in length. They are bound by members of the Argonaute protein family and play important roles in various processes including development, cellular homeostasis or response to external stimuli. Mis-regulations of miRNAs cause diseases such as cancers. In several chapters, this thesis describes miRNA profiles, miRNA and Argonaute protein characteristics as well as the de novo description of miRNAs and small RNAs in different cellular and organismal systems. The four human Argonaute (Ago) proteins are highly similar proteins. One distinct difference is the enzymatic activity of Ago2 (Meister et al., 2004), however, detailed studies on the relationship between Ago proteins have been few. This work shows that Ago2 is the most abundant Ago protein in the human cell lines HEK 293T and HeLa (Petri et al., 2011). Ago1 and Ago3 are expressed only to a minor amount, whereas Ago4 expression is hardly detectable. Re-analysis of their enzymatic activity showed that Ago2 remains the only Ago protein capable of cleaving RNA targets (Hauptmann et al., 2013). Following this lead, it could also be demonstrated that not only do Ago1 and Ago3 have difficulties in replacing the passenger strand (the strand of a small RNA duplex that usually gets degraded), but that by increasing the stability of the duplex, on-target effects of an siRNA could be elevated and off-target effects could be alleviated (Petri et al., 2011). To investigate the potential differences of the four Ago proteins with respect to their miRNA-binding properties, the distribution and the characteristics of miRNAs bound to endogenous Ago proteins were analyzed. By isolating Ago proteins using specific immunoprecipitation, cloning and sequencing of bound miRNAs, it could be shown that Ago proteins bind very similar sets of miRNAs. These miRNAs have in 10 to 20% of the cases non-templated additions of one adenosine. On the individual miRNA level, miRNAs can exist as 5’ or 3’ length variations that are called isomiRs. Investigating isomiRs, it could be shown that Ago2 binds to miRNAs with a broader isomiR spectrum than Ago1 and Ago3 (Dueck et al., 2012). The exceptional role of Ago2 is underscored in the processing of the non-canonical miR-451. Ago2 not only cleaves the precursor of miR-451 (Cheloufi et al., 2010; Cifuentes et al., 2010), but in my thesis it could also be shown that Ago2 exclusively binds miR-451 and does not exchange it with Ago1 or Ago3. Mutational studies on the miR-451 precursor highlight its unique and optimized structure, which renders its use as a backbone for Ago2-specific RNAi tools limited (Dueck et al., 2012). Following up on the analysis of individual miRNAs and their distribution among Ago proteins, global miRNA profiles under the influence of extracellular stimuli in an immunological setting were examined. Wild-type as well as miR-155-deficient macrophages and dendritic cells (DCs) were stimulated or matured with LPS, LDL, oxidized LDL (oxLDL) or enzymatically lysed LDL (eLDL). MiRNAs were cloned and sequenced and the resulting libraries were compared. Depending on the stimulus, miRNA profiles changed mildly to strongly. The use of miR-155 deficient cells proved to be a powerful tool to establish that miR-155 strongly influences miRNA expression. Examination and comparison the different global miRNA profiles highlighted that miR-155 is a master regulator of downstream miRNA expression. The green alga Volvox carteri is a model organism for the development of multicellularity. Its proteome and transcriptome is predicted to be highly similar to its closest unicellular relative, Chlamydomonas reinhardtii. Up to date, no protein(s) or mechanism(s) could be identified that explain the big difference in development and morphology between the two organisms. The small RNA repertoire of Volvox carteri and the proteins involved in their biogenesis and exertion of their function was not described when the project was started. Here, we characterized an Argonaute protein of V.carteri, AGO3, and analyzed AGO3-bound small RNAs. A miRNA identification pipeline was constructed and we identified 288 miRNAs contained in 140 miRNA families. Several of the predicted miRNAs were shown to be expressed and modified at their 3’ end, which is typical for plant miRNAs. Additionally, other small RNAs such as repeat-associated small RNAs or phased small RNAs were analyzed. Finally, no overlap of small RNA expression between Volvox and Chlamydomonas could be detected. The existence of a complete new set of small RNAs in Volvox carteri raises the question if the development of multicellularity and the division of labor between somatic and germ cells is at least partially regulated by miRNAs or small RNAs. Future studies, also on other members of the volvocine algae, need to elucidate the role of small RNAs in Volvox carteri further.