Regulation of gene expression by small RNAs
For many years transcription factors held the center stage in the regulation of gene expression. This paradigm has changed with the discovery of Piwi-protein-associated small RNAs that regulate gene expression at either transcriptional or post-transcriptional level. Among these, the microRNAs (miRNAs) have initially been discovered in the worm Caenorhabditis elegans, but in recent years they have been found in the genomes of organisms as varied as viruses, plants and humans. miRNAs play essential roles in development, metabolism, immune responses, and they can either suppress or enhance specific pathogenic processes such as infections and cancer. Although it is clear that binding of miRNA-containing RNA-induced silencing complexes to mRNAs increases the degradation rate of the mRNAs, the resulting macroscopic behaviors are still not understood. Many possibilities have been proposed, including repression of critical targets, decreasing protein noise and increasing the robustness of gene expression and providing a medium for cross-regulation between mRNAs.
Combining high-throughput experimental approaches with data analysis and computational modeling, the group of Mihaela Zavolan studies post-transcriptional regulatory circuits that control cellular differentiation. MiRNAs are an important component of these circuits, being predicted to target the majority of human genes. By modeling the combined effects of transcription factors and miRNAs on the transcriptome of various cells, the group is characterizing regulatory cascades that are triggered by miRNAs in the context of various differentiation processes. The group is especially interested to understand the dynamics of the regulatory process. A surprising finding of this work was that loading of miRNAs into the Argonaute proteins constitutes an important bottleneck that limits the speed of miRNA-dependent gene regulation. The important implication of this finding is that a rapid regulation of target expression requires active miRNA turnover in the Argonaute protein.
Fig. 2: MiRNA-dependent regulation of gene expression in single cells. HEK293 cells containing a vector encoding the green fluorescent protein and a miRNA precursor were induced with doxycycline to express these molecules. The mRNAs from individual cells are sequenced and the dependency of the miRNA target expression on GFP (and miRNA) expression was studied with computational models. The curves show that the total expression of predicted targets for two miRNAs is reduced, at the single cell level, in the presence of the miRNAs.
To further characterize the response of individual targets to miRNA expression, the group has established a cellular system in which the expression of miRNA can be progressively induced. The expression of each individual mRNA in each individual cell is measured with single cell sequencing, and mathematical models are employed to infer the parameters of interaction of each mRNA target with the miRNA.
Regulation of alternative polyadenylation
The maturation of eukaryotic mRNAs includes 5’ capping, splicing and 3’ end processing. The latter involves the endonucleolytic cleavage of the 3’ untranslated region at specific sites followed by the addition of a poly(A) tail. Ample evidence has been provided that most mammalian genes possess multiple 3’ end processing sites that can be used to generate multiple transcript isoforms through a mechanism called alternative polyadenylation (APA). Moreover, the aberrant use of poly(A) sites has been linked to proliferative states and cancer.
Pre-mRNA cleavage and polyadenylation is mediated by the 3' end processing complex, a large machinery that consists of four main subcomplexes, CF Im, CF IIm, CPSF and CstF. The action of the complex is guided by cis-regulatory elements, the best conserved of which is the so-called 'poly(A) signal', AAUAAA. By analyzing a large catalog of experimentally determined 3' end processing sites, we recently identified variants of the canonical signal, whose position-dependent frequency profiles with respect to 3' end processing sites suggest a similar function in 3' end processing.
CF Im is a subcomplex of the core 3’ end processing complex that consists of two CFIm25 and two molecules of CFIm59 and/or CFIm68 and specifically binds UGUA motifs. Previously, we have shown that knockdown of the CFIm25 and CFIm68 subunits causes a shift in polyadenylation site usage from distal to proximal sites, leading to overall shortening of 3' untranslated regions (UTRs). The physiological relevance of this mode of regulation is underscored by the recent finding that limiting CFIm25 abundance is associated with tumorigenesis (Masamha et al. Nature 510:412(2014)). APA can affect not only 3' UTR lengths, but can also occur at intronic sites as well as in exons that are close to transcription start sites (see Figure). It is critical for mRNA stability and translation and can even influence protein localization (Berkovits & Mayr Nature 522:363 (2015)). Work in our group aims to characterize the regulation of alternative poly(A) site usage and its impact for cellular behaviors.
Much of the work in the Zavolan group is collaborative. Examples of our current collaborative projects are the Sinergia project entitled “Molecular underpinnings of age-related muscle loss”, and the SystemsX.ch grants entitled “Controlling and exploiting stochasticity in gene regulatory networks” and “TargetInfectX: multi-pronged approach to pathogen infection in human cells”.
Witek Filipowicz (Friedrich Miescher Institut, Basel, CH); Helge Grosshans (Friedrich Miescher Institut, Basel, CH); Markus Stoffel (Eidgenossische Technische Hochschule Zurich, CH); Petr Svoboda (Institute of Molecular Genetics, CZ); Thomas Tuschl (The Rockefeller University, New York, US); Gunter Meister (University of Regensburg, D).