Systematic Functional Analysis of Bicaudal-D Serine Phosphorylation and Intragenic Suppression of a Female Sterile Allele of BicD

Abstract

Protein phosphorylation is involved in posttranslational control of essentially all biological processes. Using mass spectrometry, recent analyses of whole phosphoproteomes led to the identification of numerous new phosphorylation sites. However, the function of most of these sites remained unknown. We chose the Drosophila Bicaudal-D protein to estimate the importance of individual phosphorylation events. Being involved in different cellular processes, BicD is required for oocyte determination, for RNA transport during oogenesis and embryogenesis, and for photoreceptor nuclei migration in the developing eye. The numerous roles of BicD and the available evidence for functional importance of BicD phosphorylation led us to identify eight phosphorylation sites of BicD, and we tested a total of 14 identified and suspected phosphoserine residues for their functional importance in vivo in flies. Surprisingly, all these serines turned out to be dispensable for providing sufficient basal BicD activity for normal growth and development. However, in a genetically sensitized background where the BicDA40V protein variant provides only partial activity, serine 103 substitutions are not neutral anymore, but show surprising differences. The S103D substitution completely inactivates the protein, whereas S103A behaves neutral, and the S103F substitution, isolated in a genetic screen, restores BicDA40V function. Our results suggest that many BicD phosphorylation events may either be fortuitous or play a modulating function as shown for Ser103. Remarkably, amongst the Drosophila serines we found phosphorylated, Ser103 is the only one that is fully conserved in mammalian BicD.

Developmental RNA processing of 3′UTRs in Hox mRNAs as a context-dependent mechanism modulating visibility to microRNAs

“…Based on current annotated Ubx transcripts in FlyBase, our observations were unexpected; nonetheless, they are perfectly consistent with the original molecular work describing the cloning and expression of Ubx in Drosophila, which reported northern blot and other molecular and sequence data indicating the generation of Ubx mRNAs of variable 3ЈUTR length by alternative polyadenylation Kornfeld et al, 1989;O'Connor et al, 1988) (Fig. 1A).…”

“…In brief, vector pBSIIKS_mCherry-3ϫNLS (gift from Markus Affolter) (Caussinus et al, 2008) was digested with KpnI and NotI and a 837 bp fragment containing the mCherry ORF plus nuclear localisation signal (NLS) was cloned into the respective restriction sites of transformation vector pUASP.K10.attB (a gift from Beat Suter) (Koch et al, 2009). The K10 terminator sequence of resulting vector pUASP.mCherrry.3ϫNLS.K10.attB was then removed by NotI and NdeI double digestion and replaced with Ubx short (-7 to +1237 bp of annotated 3ЈUTR) or Ubx long (-7 to +2807 bp) 3ЈUTRs, which had been PCR amplified from genomic DNA.…”

“…The K10 terminator sequence of resulting vector pUASP.mCherrry.3ϫNLS.K10.attB was then removed by NotI and NdeI double digestion and replaced with Ubx short (-7 to +1237 bp of annotated 3ЈUTR) or Ubx long (-7 to +2807 bp) 3ЈUTRs, which had been PCR amplified from genomic DNA. The first polyadenylation signal [AATAAA at +950 bp of 3ЈUTR (Kornfeld et al, 1989;OЈConnor et al, 1988)] of the construct with the extended Ubx 3ЈUTR was deleted using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). To obtain transgenic flies, we used the FC31 system for site-specific integration using the ZH-attP-51C landing site (Bischof et al, 2007) (http://flyc31.frontiers-in-genetics.org/).…”

“…Information on alternative polyadenylation of Hox genes was compiled from FlyBase (http://flybase.org/) and the following references Celniker et al, 1989;Celniker et al, 1990;Kornfeld et al, 1989;Kuziora and McGinnis, 1988;Laughon et al, 1986;O'Connor et al, 1988;Rowe and Akam, 1988;Sanchez-Herrero and Crosby, 1988;Schneuwly et al, 1986;Scott et al, 1983;Stroeher et al, 1986;Tyler et al, 2008). To detect miRNA target sites, we followed methods used in previous studies (Stark et al, 2008).…”

Multiple Roles for Egalitarian in Polarization of the Drosophila Egg Chamber

“…This was done using the Q5 SiteDirected Mutagenesis Kit (New England Biolabs). Once the desired mutations were confirmed by sequencing, the entire coding sequence of eglR was cloned into pUASp-attB-K10 (Koch et al 2009). The EGFP coding sequence was subcloned immediately downstream of eglR.…”