Structural and Functional Studies of mRNA Stability Regulators

Posttranscriptional gene regulation (PTGR) is the process by which every step of the life cycle of an mRNA following transcription – maturation, transport, translation, subcellular localization and decay - is tightly regulated. This is accomplished by a complex network of multiple RNA binding proteins (RNPs) binding to several specific mRNA elements. Such cis-acting elements are or can be found within the 5’ cap, the 5’ untranslated region (UTR), the open reading frame (ORF), the 3’UTR and the poly(A) tail at the 3’ end of the mRNA. Adenylate-uridylate-rich elements (AU-rich elements; AREs) are heavily investigated regulatory cis- acting elements within 3’untranslated regions (3’UTRs). These are found in short-lived mRNAs and function as a signal for rapid degradation. AREs are present in 5-8% of human genes involved in the regulation of many essential cellular processes, such as stress response, cell cycle regulation and apoptosis and must therefore be tightly regulated. In the cytoplasm, trans-acting ARE binding proteins regulate the transport localization, stability and translation of these mRNAs. One of these factors is the embryonic lethal abnormal visual (ELAV)/ Human antigen R (HuR) protein. It increases the stability and/ or the translation of many important cellular mRNAs. Another cis-activing element is the poly(A) tail of mRNA, which protects the mRNAs from degradation. These are bound by multifunctional poly(A)-binding proteins (PABPs), which play a central role in translation initiation, translation termination and mRNA decay. In this study, we have investigated the mRNA stability regulators HuR and PABPC1, both containing multiple RNA recognition motifs (RRMs). Excitingly, some single RRMs have several functions due to the presence of additional binding interfaces that allow them to bind both RNA and other factors. We characterized the C-terminal RRM of HuR, which is hypothesized to be involved in RNA binding, homo-dimerization and protein-protein interacting. We show the first 1.9-Å-resolution crystal structure of HuR RRM3 bound to several short ARE-motifs. Our structure reveals the presence of the homodimer. The combination of several biophysical techniques validate the homo-dimerization and promiscuous RNA binding in solution. Additionally, the binding of the canonical AUUUA pentameric motifs, found in the majority of AREs, is possible by the recognition of two registers. Excitingly, RRM3 homo-dimerization increases the affinity for RNA, highlighting the cooperativity between the two binding surfaces. Moreover, despite the known stabilizing role of HuR, we provide evidence that RRM3 counteracts this effect in a Huh7 cell -based ARE reporter assay containing multiple AUUUA motifs. Finally, we investigated the mechanism of the cytoplasmic PABP RRM1 in binding to poly(A) and to the anti-proliferative B-cell translocation gene (BTG2) protein. BTG2 recruits the CCR4-associated factor 1 (CAF1), a subunit of CCR4-NOT deadenylase complex, to induce deadenylation of mRNAs. We show that PABPC1 RRM1 uses its α1 to bind BTG2 while simultaneously binding the poly(A) RNA. This interaction seems to orient the poly(A) 3’end such that it is close to the CAF1 enzymatic pocket. Our findings provide new details of the HuR RRM3-RNA recognition and homo-dimerization as well as the PABPC1 RRM1-poly(A)-BTG2 binding to recruit CAF1 and thus highlight the diversity of RRMs

AVETH Survey on Supervision of Doctoral Students

Good supervision is a key factor for the success of doctoral studies. But there are multiple good ways. Depending on the field of studies, the supervisor ‘style’, and the students’ specific needs, multiple approaches can lead to good results. This variety of supervision contexts makes it difficult to have an overview of the supervision practices, and their actual impact on students’ satisfaction. Thus, in fall 2017, AVETH conducted a survey on the doctoral supervision practices at ETH Zurich, with two objectives: (i) draw a picture of the actual supervision practices at ETH, and (ii) investigate the doctoral students’ satisfaction with respect to their supervision and the impact of specific practices. Based on 1’594 completed survey answers (corresponding to a response rate of ~36%) this report summarizes the findings. It appears that 62% of doctoral students are generally satisfied (Grade 6 and above – See figure below) and 40% are very satisfied (Grade 8 and above) with their supervision. However these numbers vary a lot across departments. Furthermore, there are relations between satisfaction and (i) the number of years of the doctoral thesis, (ii) the opportunities for scientific interactions (both within and outside of the group), and (iii) formal or informal appraisal interviews. Finally, 24% of the survey respondents stated that they experience some kind of ‘abuse of power’ from their supervisor, ranging from lack of scientific freedom to pressure to work long hours or on weekends. This survey offers a factual description of supervision practices at ETH Zurich and raises some alert flags on practices, which should be monitored and/or prevented. Using this new information, AVETH will work together with the ETH School Board to propose a set of ‘supervision guidelines’, which will hopefully contribute to improve everyone’s situation at ETH

Aromatic side-chain conformational switch on the surface of the RNA Recognition Motif enables RNA discrimination

The cyclooxygenase-2 is a pro-inflammatory and cancer marker, whose mRNA stability and translation is regulated by the CUG-binding protein 2 interacting with AU-rich sequences in the 3′ untranslated region. Here, we present the solution NMR structure of CUG-binding protein 2 RRM3 in complex with 5′-UUUAA-3′ originating from the COX-2 3′-UTR. We show that RRM3 uses the same binding surface and protein moieties to interact with AU- and UG-rich RNA motifs, binding with low and high affinity, respectively. Using NMR spectroscopy, isothermal titration calorimetry and molecular dynamics simulations, we demonstrate that distinct sub-states characterized by different aromatic side-chain conformations at the RNA-binding surface allow for high- or low-affinity binding with functional implications. This study highlights a mechanism for RNA discrimination possibly common to multiple RRMs as several prominent members display a similar rearrangement of aromatic residues upon binding their targets.ISSN:2041-172

Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate–rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3′ untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.ISSN:0027-8424ISSN:1091-649

RNase L activation in the cytoplasm induces aberrant processing of mRNAs in the nucleus.

The antiviral endoribonuclease, RNase L, is activated by the mammalian innate immune response to destroy host and viral RNA to ultimately reduce viral gene expression. Herein, we show that RNase L and RNase L-mediated mRNA decay are primarily localized to the cytoplasm. Consequently, RNA-binding proteins (RBPs) translocate from the cytoplasm to the nucleus upon RNase L activation due to the presence of intact nuclear RNA. The re-localization of RBPs to the nucleus coincides with global alterations to RNA processing in the nucleus. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and correlate with their retention in the nucleus and reduction in interferon protein production. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is dictated by the availability of RNA in each compartment. Thus, viral infections that trigger RNase L-mediated cytoplasmic RNA in the cytoplasm also alter RNA processing in the nucleus, resulting in an ingenious multi-step immune block to protein biogenesis

Characterization of Molecular Interactions between ACP and Halogenase Domains in the Curacin A Polyketide Synthase

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are large multidomain proteins present in microorganisms that produce bioactive compounds. Curacin A is such a bioactive compound with potent anti-proliferative activity. During its biosynthesis the growing substrate is bound covalently to an acyl carrier protein (ACP) that is able to access catalytic sites of neighboring domains for chain elongation and modification. While ACP domains usually occur as monomers, the curacin A cluster codes for a triplet ACP (ACP_I-ACP_II-ACP_III) within the CurA PKS module. We have determined the structure of the isolated holo-ACP_I and show that the ACPs are independent of each other within this tridomain system. In addition, we have determined the structure of the 3-hydroxyl-3-methylglutaryl-loaded holo-ACP_I, which is the substrate for the unique halogenase (Hal) domain embedded within the CurA module. We have identified the interaction surface of both proteins using mutagenesis and MALDI-based identification of product formation. Amino acids affecting product formation are located on helices II and III of ACP_I and form a contiguous surface. Since the CurA Hal accepts substrate only when presented by one of the ACPs within the ACP_I-ACP_II-ACP_III tridomain, our data provide insight into the specificity of the chlorination reaction