The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization

<p>Abstract</p> <p>Background</p> <p>Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD) is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants.</p> <p>Results</p> <p>The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1) RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus.</p> <p>Conclusions</p> <p>The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to date. This, along with the amino acid variability observed within their PUM-HDs, suggests that these proteins may be involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions and throughout development.</p

Flipped design for learning: Deepening scientific inquiry in a large-enrollment class

Hypothesis testing is central not only to the scientific method but also to understanding the nature of scientific knowledge. Although it is widely appreciated that students should develop hypothesis testing skills early in their undergraduate careers, there are many challenges in large-enrollment classes that can prevent them from deeply understanding the process of scientific inquiry. The hypothesis of this study is that a flipped-learning design will create a more effective environment than a traditional lecture format in which to foster both content acquisition and an understanding of the process of scientific inquiry. To measure the relative impact of these two approaches on learning, our study compares cohorts of students in different sections of the same large-enrollment course who have been exposed either to a flipped design (combining collaborative in-class activities with problem-based computer simulation software) or a lecture-based approach. The key principles underlying our research design as well as preliminary findings of pre- and post-assessment surveys measuring student understanding of scientific inquiry and basic content acquisition will be presented. An initial analysis of the data obtained from focus groups will also be discussed. Although the project is still at an early stage, preliminary data suggest that a combination of peer-learning in-class activities and problem-based computer simulation software foster both the acquisition of content and the development of scientific inquiry skills

Program SAGES: Promoting collaborative teaching development through graduate student/faculty partnerships

Each year, graduate students shoulder hours of instructional time with undergraduate students and some have more contact hours with students than academic staff in large introductory undergraduate courses. However, many graduate students are given minimal opportunities for teaching development, and there is a great need to help them develop a scholarly and reflective teaching practice (Kenny et al., 2014; Chick & Brame, 2015). Enhancing the teaching skills of graduate students is a critical investment that will also create a culture of educational leadership, and foster innovation and teaching development. To support STEM graduate students in the development of an evidence-based teaching practice, we designed and implemented the SAGES Program (SoTL Advancing Graduate Education in STEM) at a research-intensive university. This program was designed to provide graduate students with opportunities to learn about scholarly teaching and learning (SoTL) within the context of STEM through a semester-long course, followed by a semester-long practicum. The practicum gives graduate students an opportunity to apply their learning in an undergraduate class, in partnership with a faculty member acting as a mentor. Through a mixed-methods approach based on the use of semi-structured interviews and pretest and posttest surveys (DeChenne et al., 2012; Trigwell and Prosser, 2004), we will show that SAGES not only increased teaching self-efficacy, knowledge and skills in graduate students, but also led to collaborative teaching development for both mentors and mentees. We will also invite participants to conceptualize how such a program could be designed for their own institutions. Chick, N. L. & Brame, C. (2015). An investigation of the products and impact of graduate student SoTL programs: observations and recommendations from a single institution. International Journal for the Scholarship of Teaching and Learning, 9(1), article 3. DeChenne, S. E., Enochs, L. G., & Needham, M. (2012) Science, technology, engineering, and mathematics graduate teaching assistants teaching self-efficacy. Journal of the Scholarship of Teaching and Learning, 12(4), 102-123. Kenny, N., Watson, G. P. L., & Walton, C. (2014) Exploring the context of Canadian graduate student teaching certificates in university teaching. Canadian Journal of Higher Education, 44(3), 1-19. Trigwell, K. & Prosser, M. (2004) Development and use of the approaches to teaching inventory. Educational Psychology, 16(4), 409-424

A second, non-canonical RNA-dependent RNA polymerase in SARS Coronavirus

In (+) RNA coronaviruses, replication and transcription of the giant ∼30 kb genome to produce genome- and subgenome-size RNAs of both polarities are mediated by a cognate membrane-bound enzymatic complex. Its RNA-dependent RNA polymerase (RdRp) activity appears to be supplied by non-structural protein 12 (nsp12) that includes an RdRp domain conserved in all RNA viruses. Using SARS coronavirus, we now show that coronaviruses uniquely encode a second RdRp residing in nsp8. This protein strongly prefers the internal 5′-(G/U)CC-3′ trinucleotides on RNA templates to initiate the synthesis of complementary oligonucleotides of <6 residues in a reaction whose fidelity is relatively low. Distant structural homology between the C-terminal domain of nsp8 and the catalytic palm subdomain of RdRps of RNA viruses suggests a common origin of the two coronavirus RdRps, which however may have evolved different sets of catalytic residues. A parallel between the nsp8 RdRp and cellular DNA-dependent RNA primases is drawn to propose that the nsp8 RdRp produces primers utilized by the primer-dependent nsp12 RdRp