Supporting Biomedical Research Training for Historically Underrepresented Undergraduates Using Interprofessional, Nonformal Education Structures.

Research experience provides critical training for new biomedical research scientists. Students from underrepresented populations studying science, technology, engineering, and mathematics (STEM) are increasingly recruited into research pathways to diversify STEM fields. However, support structures outside of research settings designed to help these students navigate biomedical research pathways are not always available; nor are program support components outside the context of laboratory technical skills training and formal mentorship well understood. This study leveraged a multi-institutional research training program, Enhancing Cross-Disciplinary Infrastructure and Training at Oregon (EXITO), to explore how nine institutions designed a new curricular structure (Enrichment) to meet a common goal of enhancing undergraduate research training and student success. EXITO undergraduates participated in a comprehensive, 3-year research training program with the Enrichment component offered across nine sites: three universities and six community colleges, highly diverse in size, demographics, and location. Sites' approaches to supporting students in the training program were studied over a 30-month period. All sites independently created their own nonformal curricular structures, implemented interprofessionally via facilitated peer groups. Site data describing design and implementation were thematically coded to identify essential programmatic components across sites, with student feedback used to triangulate findings. Enrichment offered students time to critically reflect on their interests, experiences, and identities in research; network with peers and professionals; and support negotiation of hidden and implicit curricula. Students reported the low-pressure setting and student-centered curriculum balanced the high demands associated with academics and research. Core curricular themes described Enrichment as fostering a sense of community among students, exposing students to career paths and skills, and supporting development of students' professional identities. The non-formal, interprofessional curricula enabled students to model diverse biomedical identities and pathways for each other while informing institutional structures to improve diverse undergraduate students' success in academia and research.

Arginine methylation of RNA-binding proteins regulates cell function and differentiation.

Arginine methylation is a post-translational modification that regulates protein function. RNA-binding proteins are an important class of cell-function mediators, some of which are methylated on arginine. Early studies of RNA-binding proteins and arginine methylation are briefly introduced, and the enzymes that mediate this post-translational modification are described. We review the most common RNA-binding domains and briefly discuss how they associate with RNAs. We address the following groups of RNA-binding proteins: hnRNP, Sm, Piwi, Vasa, FMRP, and HuD. hnRNPs were the first RNA-binding proteins found to be methylated on arginine. The Sm proteins function in RNA processing and germ cell specification. The Piwi proteins are largely germ cell specific and are also required for germ cell production, as is Vasa. FMRP participates in germ cell formation in Drosophila, but is more widely known for its neuronal function. Similarly, HuD plays a role in nervous system development and function. We review the effects of arginine methylation on the function of each protein, then conclude by addressing remaining questions and future directions of arginine methylation as an important and emerging area of regulation.

A new regulatory function of the region proximal to the RGG box in the fragile X mental retardation protein.

Fragile X mental retardation protein (FMRP) is required for normal cognition. FMRP has two autosomal paralogs, which although similar to FMRP, cannot compensate for the loss of FMRP expression in brain. The arginine- and glycine-rich region of FMRP (the RGG box) is unique; it is the high-affinity RNA-binding motif in FMRP and is encoded by exon 15. Alternative splicing occurs in the 5' end of exon 15, which is predicted to affect the structure of the distally encoded RGG box. Here, we provide evidence that isoform 3, which removes 25 amino acids from the 5' end of exon 15, has an altered conformation that reduces binding of a specific antibody and renders the RGG box unable to efficiently associate with polyribosomes. Isoform 3 is also compromised in its ability to form granules and to associate with a key messenger ribonucleoprotein Yb1 (also known as p50, NSEP1 and YBX1). Significantly, these functions are similarly compromised when the RGG box is absent from FMRP, suggesting an important regulatory role of the N-terminal region encoded by exon 15.

Fragile X protein family member FXR1P is regulated by microRNAs.

FXR1P is one of two autosomal paralogs of the fragile X mental retardation protein FMRP. The absence of FMRP causes fragile X syndrome, the leading cause of hereditary mental retardation. FXR1P plays an important role in normal muscle development and has been implicated in facioscapulohumeral muscular dystrophy (FSHD). Its absence also causes cardiac abnormalities in both mice and zebrafish. To examine miRNA-mediated regulation of FMRP and FXR1P, we studied their expression in a conditional Dicer knockdown cell line, DT40. We found that FXR1P, but not FMRP, is significantly increased upon Dicer knockdown and the consequent reduction of miRNAs, suggesting that FXR1P is regulated by miRNAs while FMRP is not in DT40 cells. Expression of a luciferase reporter bearing the 3' untranslated region (3'UTR) of FXR1 was significantly increased in the absence of miRNAs, confirming miRNA-mediated regulation of FXR1P, while a luciferase reporter bearing the FMR1 3'UTR was not. We identified one of the regulatory regions in the 3'UTR of FXR1 by removing a conserved, 8-nucleotide miRNA seed sequence common to miRNAs 25, 32, 92, 363, and 367 and demonstrated loss of miRNA-mediated suppression. Treatment with specific miRNA hairpin inhibitors to each of the miRNAs in the seed sequence showed that miRs 92b, 363, and 367 regulated FXR1P expression. Accordingly, overexpression of the miRNA 367 mimic significantly decreased endogenous FXR1P expression in human cell lines HEK-293T and HeLa. We report for the first time that FXR1P is regulated through miRNA binding, with one site being the miR-25/32/92/363/367 seed sequence.

Arginines of the RGG box regulate FMRP association with polyribosomes and mRNA.

Fragile X syndrome is caused by the loss of expression of the fragile X mental retardation protein, FMRP. FMRP is an RNA-binding protein that is highly expressed in neurons and undergoes multiple post-translational modifications including methylation on arginine. FMRP is methylated on the high-affinity RNA-binding motif, the RGG box, at positions 533, 538, 543 and 545 of murine FMRP. To identify the arginines important for FMRP function, we examined their role in polyribosome and mRNA association. We found that arginines 533 and 538 were required for normal FMRP polyribosome association whereas all four arginines played a role in RNA binding, depending on the identity of the RNA. The model G-quadruplex RNA sc1 required arginines 533 and 538 for normal association with FMRP, whereas AATYK mRNA did not. In vitro methylation of FMRP-bearing arginine substitutions inhibited sc1 binding but not AATYK binding. In addition, we found that PRMT1 co-immunoprecipitated with FMRP isolated from cells and that siRNAs directed against PRMT1 led to reduced FMRP methylation. Thus, two lines of experimentation demonstrate that PRMT1 acts on FMRP in cells. In summary, we provide evidence for the important role of the RGG box in polyribosome association. We also demonstrate for the first time that the different arginines of the RGG box are important for the binding of different RNAs. Finally, we show that PRMT1 methylates FMRP in cells, suggesting a model where methylation of the RGG box modulates either the quantity or the identity of the RNAs bound by FMRP.

The pheromone-induced nuclear accumulation of the Fus3 MAPK in yeast depends on its phosphorylation state and on Dig1 and Dig2.

BACKGROUND: Like mammalian MAP kinases, the mating-specific Fus3 MAPK of yeast accumulates in the nuclei of stimulated cells. Because Fus3 does not appear to be subjected to active nucleo-cytoplasmic transport, it is not clear how its activation by mating pheromone effects the observed change in its localization. One possibility is that the activation of Fus3 changes its affinity for nuclear and cytoplasmic tethers. RESULTS: Dig1, Dig2, and Ste12 are nuclear proteins that interact with Fus3. We found that the pheromone-induced nuclear accumulation of a Fus3-GFP reporter is reduced in cells lacking Dig1 or Dig2, whereas Fus3T180AY182A-GFP localization was unaffected by the absence of these proteins. This suggests that Dig1 and Dig2 contribute to the retention of phosphorylated Fus3 in the nucleus. Moreover, overexpression of Ste12 caused the hyper-accumulation of Fus3-GFP (but not Fus3T180AY182A-GFP) in the nuclei of pheromone-treated cells, suggesting that Ste12 also plays a role in the nuclear retention of phosphorylated Fus3, either by directly interacting with it or by transcribing genes whose protein products are Fus3 tethers. We have previously reported that overexpression of the Msg5 phosphatase inhibits the nuclear localization of Fus3. Here we show that this effect depends on the phosphatase activity of Msg5, and provide evidence that both nuclear and cytoplasmic Msg5 can affect the localization of Fus3. CONCLUSION: Our data are consistent with a model in which the pheromone-induced phosphorylation of Fus3 increases its affinity for nuclear tethers, which contributes to its nuclear accumulation and is antagonized by Msg5.

Effect of the pheromone-responsive G(alpha) and phosphatase proteins of Saccharomyces cerevisiae on the subcellular localization of the Fus3 mitogen-activated protein kinase.

The mating-specific G(alpha) protein of Saccharomyces cerevisiae, Gpa1, stimulates adaptation to pheromone by a mechanism independent of G(beta gamma) sequestration. Genetic evidence suggests that Gpa1 targets the Fus3 mitogen-activated protein kinase, and it has recently been shown that the two proteins interact in cells responding to pheromone. To test the possibility that Gpa1 downregulates the mating signal by affecting the localization of Fus3, we created a Fus3-green fluorescent protein (GFP) fusion protein. In vegetative cells, Fus3-GFP was found in both the cytoplasm and the nucleus. Pheromone stimulated a measurable increase in the ratio of nuclear to cytoplasmic Fus3-GFP. In contrast, the relative level of nuclear Fus3-GFP decreased as cells recovered from pheromone arrest and did not increase when cells adapted to chronic stimulus were challenged again. Accumulation of Fus3-GFP in the nuclei of stimulated cells was also inhibited by overexpression of either wild-type Gpa1, the E364K hyperadaptive mutant form of Gpa1, or the Msg5 dually specific phosphatase. The effects of Gpa1 and Msg5 on Fus3 are partially interdependent. In a genetic screen for adaptive defective mutants, a nonsense allele of the nucleocytoplasmic transport receptor, Kap104, was identified. Truncation of the Kap104 cargo-binding domain blocked the effect of both Gpa1(E364K) and Msg5 on Fus3-GFP localization. Based on these results, we propose that Gpa1 and Msg5 work in concert to downregulate the mating signal and that they do so by inhibiting the pheromone-induced increase of Fus3 in the nucleus. Kap104 is required for the G(alpha)/phosphatase-mediated effect on Fus3 localization.