Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins

In the last 15years substantial advances have been made to place isotope labels in native and glycosylated proteins for NMR studies and structure determination. Key developments include segmental isotope labeling using Native Chemical Ligation, Expressed Protein Ligation and Protein Trans-Splicing. These advances are pushing the size limit of NMR spectroscopy further making larger proteins accessible for this technique. It is just emerging that segmental isotope labeling can be used to define inter-domain interactions in NMR structure determination. Labeling of post-translational modified proteins like glycoproteins remains difficult but some promising developments were recently achieved. Key achievements are segmental and site-specific labeling schemes that improve resonance assignment and structure determination of the glycan moiety. We adjusted the focus of this perspective article to concentrate on the NMR applications based on recent developments rather than on labeling methods themselves to illustrate the considerable potential for biomolecular NM

Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte

A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals

Medical Education Escape Room Aligned with Flipped Classroom and Powered by Mobile Augmented Reality

Medical education escape rooms are emerging as a viable technological resource for pedagogy-first, learner-centric educational activities. This work presents the evaluation results of the first flipped classroom implementation in medical education, thus utilizing a mobile-driven augmented reality (AR) escape room. A total of 21 first-year medical students attended a flipped classroom educational activity that aimed to acclimate the students with the workflows of basic life support. Knowledge acquisition and user perceptions were evaluated. Knowledge acquisition was evaluated with an ad hoc relevant instrument at three timepoints: (a) baseline at recruitment, (b) preclass after students had prepared for the episode, and (c) after class. Learner perceptions about the activity and the AR escape room were recorded at the activity’s end using a previously designed evaluation instrument. The results demonstrated sufficient knowledge acquisition only after completing the whole educational activity, while learners found the experience interesting, and the AR escape room challenging, thus reflecting an activity that was well formulated in structure and content. The challenges identified were the limited out of class collaboration capacity of the digital application and the highly gamified approach that at points counteracted the educational scope of the activity. Overall, these positive initial results demonstrate the potential of collaborative, escape based, activities for self-directed, learner-centric medical education

A novel occluded RNA recognition motif in Prp24 unwinds the U6 RNA internal stem loop

The essential splicing factor Prp24 contains four RNA Recognition Motif (RRM) domains, and functions to anneal U6 and U4 RNAs during spliceosome assembly. Here, we report the structure and characterization of the C-terminal RRM4. This domain adopts a novel non-canonical RRM fold with two additional flanking α-helices that occlude its β-sheet face, forming an occluded RRM (oRRM) domain. The flanking helices form a large electropositive surface. oRRM4 binds to and unwinds the U6 internal stem loop (U6 ISL), a stable helix that must be unwound during U4/U6 assembly. NMR data indicate that the process starts with the terminal base pairs of the helix and proceeds toward the loop. We propose a mechanistic and structural model of Prp24′s annealing activity in which oRRM4 functions to destabilize the U6 ISL during U4/U6 assembly

Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein

Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3′ oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3′ UUUOH trailer. An example of the latter is the binding of La to the IRES domain IV of the hepatitis C virus (HCV) RNA, which is associated with viral translation stimulation. By systematic biophysical investigations, we have found that La binds to domain IV using an RNA recognition that is quite distinct from its mode of binding to RNAs with a 3′ UUUOH trailer: although the La motif and first RNA recognition motif (RRM1) are sufficient for high-affinity binding to 3′ oligoU, recognition of HCV domain IV requires the La motif and RRM1 to work in concert with the atypical RRM2 which has not previously been shown to have a significant role in RNA binding. This new mode of binding does not appear sequence specific, but recognizes structural features of the RNA, in particular a double-stranded stem flanked by single-stranded extensions. These findings pave the way for a better understanding of the role of La in viral translation initiation

Modular protein-RNA interactions regulating mRNA metabolism: a role for NMR

Here we review the role played by transient interactions between multi-functional proteins and their RNA targets in the regulation of mRNA metabolism, and we describe the important function of NMR spectroscopy in the study of these systems. We place emphasis on a general approach for the study of different features of modular multi-domain recognition that uses well-established NMR techniques and that has provided important advances in the general understanding of post-transcriptional regulation

The Germ Cell Nuclear Proteins hnRNP G-T and RBMY Activate a Testis-Specific Exon

The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2β is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5′ splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2β and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3′ splice site normally weakly selected in the testis. Co-expression of Tra2β with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein

Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition

c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcription of this oncogene is governed by the counterbalancing forces of two proteins on TFIIH—the FUSE binding protein (FBP) and the FBP-interacting repressor (FIR). FBP and FIR recognize single-stranded DNA upstream of the P1 promoter, known as FUSE, and influence transcription by oppositely regulating TFIIH at the promoter site. Size exclusion chromatography coupled with light scattering reveals that an FIR dimer binds one molecule of single-stranded DNA. The crystal structure confirms that FIR binds FUSE as a dimer, and only the N-terminal RRM domain participates in nucleic acid recognition. Site-directed mutations of conserved residues in the first RRM domain reduce FIR's affinity for FUSE, while analogous mutations in the second RRM domain either destabilize the protein or have no effect on DNA binding. Oppositely oriented DNA on parallel binding sites of the FIR dimer results in spooling of a single strand of bound DNA, and suggests a mechanism for c-myc transcriptional control