Biomedical subjects

S Grünert

Publications and source records attributed to S Grünert.

6 recordsLinked to original sources

PubMed2000

Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation.

Drosophila Staufen protein is required for the localization of oskar mRNA to the posterior of the oocyte, the anterior anchoring of bicoid mRNA and the basal localization of prospero mRNA in dividing neuroblasts. The only regions of Staufen that have been conserved throughout animal evolution are five double-stranded (ds)RNA-binding domains (dsRBDs) and a short region within an insertion that splits dsRBD2 into two halves. dsRBDs 1, 3 and 4 bind dsRNA in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length Staufen protein lacking this insertion is able to associate with oskar mRNA and activate its translation, but fails to localize the RNA to the posterior. In contrast, Staufen lacking dsRBD5 localizes oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the microtubule-dependent localization of osk mRNA, and dsRBD5 for the derepression of oskar mRNA translation, once localized. Since dsRBD5 has been shown to direct the actin-dependent localization of prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based mRNA transport.

Actins↗

PubMed2000

RNA recognition by a Staufen double-stranded RNA-binding domain.

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem-loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.

Amino Acid Sequence↗

PubMed1996

RNA localization and the development of asymmetry during Drosophila oogenesis.

Recent work on axis formation in Drosophila has revealed that polarity arises in several distinct stages during oogenesis. One cell of a germline cyst is selected to become the oocyte, the position of the oocyte determines the posterior of the follicle, and the position of the oocyte nucleus determines the dorsal side. Each of these symmetry-breaking steps involves the asymmetric localization of a unique structure, leading to polarization of the cytoskeleton and the localization of specific mRNAs.

Animals↗

PubMed1995

NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5.

The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR. Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet. This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way.

Amino Acid Sequence↗

PubMed1994

The immediate downstream codon strongly influences the efficiency of utilization of eukaryotic translation initiation codons.

Nucleotide substitutions were introduced into the initiation site of an influenza virus NS cDNA derivative at the +4, +5 and +6 positions (where the A of the AUG codon is defined as +1), in the background of either AUG or CUG as the initiation codon. Capped transcripts of these constructs were translated in rabbit reticulocyte lysate under conditions where the selection of initiation sites conformed to the scanning ribosome model. With CUG as the initiation codon, the efficiency of initiation was as strongly influenced by the nature of the residue in the +5 position as at +4, whilst the influence of the +6 position was smaller. The residues favourable to initiation were as follows: at +4, only G was stimulatory; at +5, A was strongly stimulatory and C fairly beneficial; and at +6, only U exerted any positive influence. The positive influence of the favourable residues (or the negative influence of unfavourable residues) at each position appeared to be additive. With AUG as the initiation codon, the pattern of response to mutations in the +4 and +5 positions was qualitatively similar, but the quantitative effects were smaller. Thus the optimum downstream context for initiation is A/CUGGAU.

Base Sequence↗

PubMed1993

Substitution mutations at the putative catalytic triad of the poliovirus 3C protease have differential effects on cleavage at different sites.

Picornavirus 3C proteases are substrate-specific cysteine proteases, proposed to be homologous to the trypsin/chymotrypsin-like serine proteases on the basis of structural predictions. Substitutions at the putative active-site residues (Glu71 and Cys147) of the poliovirus 3C protease did not completely abolish proteolytic processing in vitro. The activity of mutated 3C proteases was in the following hierarchy: Glu71-Cys147 (wild type) > Asp71-Cys147 > Glu71-Ser147 > Gln71-Cys147 > Asp71-Ser147 > Gln71-Ser147 (inactive at all sites). Such mutations had differential effects on cleavage at different sites of the poliovirus polyprotein. Cleavage within the P1 region of the polyprotein was the most defective, at the 1ABC/VP1 junction and particularly at the VP0/VP3 junction. Cleavage at the 3AB/3CD and 2B/2C junctions was less affected by the mutations, and the P2/P3 and 2A/2BC junctions were cleaved efficiently by all mutants except Gln71-Ser147. All the 3C mutants gave negative results in infectivity and replication assays after transfection, indicating that mutation of Glu71 or Cys147 virtually abolishes viral replication, irrespective of the efficiency of processing of the nonstructural part of the polyprotein.

3C Viral Proteases↗