A proteomic investigation of soluble olfactory proteins in Anopheles gambiae

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector. © 2013 Mastrobuoni et al

Analysis of Intron Sequences Reveals Hallmarks of Circular RNA Biogenesis in Animals

Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human ALU repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched in comparison to linear controls. By scoring the presence of reverse complementary sequences in human introns, we predicted and experimentally validated circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyperediting events. Knockdown of the double-strand RNA-editing enzyme ADAR1 significantly and specifically upregulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA-RNA interactions of introns form larger structures that promote circularization of embedded exons, whereas ADAR1 antagonizes circRNA expression by melting stems within these interactions

A proteomic investigation of soluble olfactory proteins in Anopheles gambiae.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector

Conserved functional antagonism of CELF and MBNL proteins controls stem cell-specific alternative splicing in planarians

In contrast to transcriptional regulation, the function of alternative splicing (AS) in stem cells is poorly understood. In mammals, MBNL proteins negatively regulate an exon program specific of embryonic stem cells; however, little is known about the in vivo significance of this regulation. We studied AS in a powerful in vivo model for stem cell biology, the planarian Schmidtea mediterranea. We discover a conserved AS program comprising hundreds of alternative exons, microexons and introns that is differentially regulated in planarian stem cells, and comprehensively identify its regulators. We show that functional antagonism between CELF and MBNL factors directly controls stem cell-specific AS in planarians, placing the origin of this regulatory mechanism at the base of Bilaterians. Knockdown of CELF or MBNL factors lead to abnormal regenerative capacities by affecting self-renewal and differentiation sets of genes, respectively. These results highlight the importance of AS interactions in stem cell regulation across metazoans.We acknowledge support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208 and BFU2014-55076-P to MI and a CIHR Operating Grant MOP-125894 to QDM and TRH

Western-blot of crude antennal extracts of male and female <i>An. gambiae</i>, using polyclonal antisera against OBPs 9, 4 and 5.

<p>Left panels: SDS-PAGE of crude extracts (Ex) and sample of purified OBPs as indicated by their numbers. Right panels: Western-blot analysis of crude extracts (Ex) performed with the three antisera. A sample of OBPs 9, 4 and 5 (0.5 µg of each protein) utilised for raising the antibodies was also loaded on the same gel. OBP4 and 5 are not detectable in our experimental conditions, while OBP9 is present in both sexes, in agreement with the shotgun experiment results. Molecular weight markers are, from the top: Bovine serum albumin (66 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (29 kDa), Trypsin inhibitor (20 kDa), α-Lactalbumin (14 kDa).</p

Expression of <i>An. gambiae</i> OBP5 in <i>E. coli</i>. SDS-PAGE of bacterial pellets before (Pre) and after (Ind) induction of the culture with Isopropyl-1-thio-β-D-galacto-pyranoside.

<p>Molecular weight markers are, from the top: Bovine serum albumin (66 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (29 kDa), Trypsin inhibitor (20 kDa), α-Lactalbumin (14 kDa). OBP5: purified protein.</p

Abundance of OBPs, CSPs and other proteins in the antennae of <i>An. gambiae</i> males and females, as reported in Table 1.

<p>The evaluation of relative abundance (in arbitrary units) is based on the values produced by MaxQuant (see text). The values are the averages of three sets of analyses. Error bars represent standard error of the mean. By far the most abundant proteins in male antennae are OBP9, SAP1 and SAP3, in agreement with the results of the 2D-gel (Figure 1).</p

Two-dimensional gel electrophoretic separation of extracts from 100 fourth instar larvae and 100 pupae of <i>An. gambiae</i>.

<p>The gel was stained with colloidal Coomassie Brilliant Blue and all the spots migrating with apparent molecular weight lower than 24(coverage by aminoacid sequence up to 61.87%), found in several spots (red circles). In larvae we could also detect OBP21 (Entry code in Uniprot Q8I8S3; coverage by aminoacid sequence 9.16%) and SAP3 (coverage by aminoacid sequence up to 18.25%), present in spots where also OBP9 was identified. Molecular weight markers are, from the top: Phosphorylase b, from rabbit muscle (97 kDa), Bovine serum albumin (66 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (29 kDa), Trypsin inhibitor (20 kDa), α-Lactalbumin (14 kDa).</p

Two-dimensional gel electrophoretic separation of an extract from 1,100 antennae of <i>An. gambiae</i>.

<p>The gel was stained with colloidal Coomassie Brilliant Blue and all the spots migrating with apparent molecular weight lower than 40: Phosphorylase b, from rabbit muscle (97 kDa), Bovine serum albumin (66 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (29 kDa), Trypsin inhibitor (20 kDa), α-Lactalbumin (14 kDa).</p