#WirVsVirus: Communities co-producing new solutions to meet Covid-19 challenges through a hackathon in Germany

This chapter provides a conceptual framework for digital co-production, including co-commissioning, co-design, codelivery, and co-assessment of public services and outcomes. It illustrates this conceptual framework through a German case study involving citizens participating in a large-scale hackathon to meet COVID-19 challenges. In particular, we will analyse how the #WirVsVirus hackathon (https:// wirvsvirus.org/hackaton/) put co-production into practice from the perspective of a hackathon participant (Claudia Masiga), who has been working on the OpenFoodBank initiative

Proteins associated with SF3a60 in T. brucei.

Trypanosoma brucei relies on Spliced leader trans splicing to generate functional messenger RNAs. Trans splicing joins the specialized SL exon from the SL RNA to pre-mRNAs and is mediated by the trans-spliceosome, which is made up of small nuclear ribonucleoprotein particles and non-snRNP factors. Although the trans spliceosome is essential for trypanosomatid gene expression, not all spliceosomal protein factors are known and of these, only a few are completely characterized. In this study, we have characterized the trypanosome Splicing Factor, SF3a60, the only currently annotated SF3a component. As expected, epitope-tagged SF3a60 localizes in the trypanosome nucleus. SF3a60 is essential for cell viability but its depletion seem to have no detectable effect on trans-splicing. In addition, we used SF3a60 as bait in a Yeast-2-hybrid system screen and identified its interacting protein factors. The interactions with SF3a120, SF3a66 and SAP130 were confirmed by tandem affinity purification and mass spectrometry

S3a60 is essential for trypanosome viability.

<p>(A) Cumulative growth curves of TbSF3a60 depleted trypanosomes. The cells grown with (triangles, solid line) or without (squares, dashed line) tetracycline. Every 24 h, samples were taken for the determination of cell density (cells/ml), and grown cultures were diluted down to 5×10⁵ cells/ml with fresh medium. (B). Effect on <i>SF3a60</i> mRNA. Cells were grown either without <i>Tet</i> (–), or in the presence of 100 ng/ml <i>Tet</i> (+) for 24, 48 and 72 hours. Each lane on the Northern blot contains 10 μg RNA from bloodstream form wild type (WT) or an RNAi cell line. Asterisk (*) denotes dsRNA. Trypanosome SRP and Tubulin (TUB) RNAs are used as loading controls. Arrow indicates SF3a60 mRNA.</p

SF3a60-associated protein preys from a genome-wide Yeast-2-hybrid screen.

1<p>Confidence levels according to PBS scores <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091956#pone.0091956-Formstecher1" target="_blank">[32]</a>.</p>2<p>Domains (superfamily, Pfam) predicted in TritrypDB (<a href="http://tritrypdb.org/tritrypdb/" target="_blank">http://tritrypdb.org/tritrypdb/</a>); a blank space indicates those without known domains.</p>3<p>Functional designation. Functions predicted on the basis of sequence match alone are shown in parentheses.</p

SF3a60 RNAi has no effect on splicing.

<p>The Y structure <i>trans</i> splicing intermediate is not decreased after two days of RNAi targeting SF3a60. The full-length spliced leader RNA and the Y structure were detected by primer extension (diagram in panel A) followed by denaturing polyacrylamide gel electrophoresis (B) with the U3 snRNA as a loading control. The RNA was prepared from the RNAi cell line grown without tetracycline (panel B, lanes 1 and 4), or with tetracycline for one or two days (lanes 2 and 3). M: markers.</p

Proteins associated with SF3a60 by tandem affinity purification.

<p>Proteins associated with SF3a60 by tandem affinity purification.</p

Preparative tandem affinity purification identifies SF3a60 associated proteins.

<p>Proteins were separated on 12% SDS-PAGE and stained with SYPRO Ruby. Marker protein sizes in kilo Daltons are indicated on the left and corresponding protein bands are on the right. Details are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091956#pone-0091956-t001" target="_blank">Table 1</a>.</p

Sequence conservation for putative T. brucei PRP40.

<p><b>A</b>. The WW domains of human FBP11 (O75400.2), yeast PRP40 (NP_012913.3) and the putative Trypanosome FBP11 homologue contain the tryptophans, prolines and a central set of three hydrophobic residues characteristic of WW domains. Evidently, the putative <i>T. brucei</i> FBP11 lacks a second WW domain.</p

Trypanosome SF3a60 localizes in the nucleus.

<p>(A) Cells expressing <i>SF3a60</i>-TAP and the TAP tag with (+) and without (−) tetracycline induction after Western blot analyses. The SF3a60-TAP is conditionally expressed while tubulin (TUB) expression is used as control and (B). Immunofluorescence of procyclic form trypanosomes TAP-tagged <i>TbSF3a60</i> counterstained for <i>Tb</i>SF3a60 (red) and for DNA (DAPI).</p