Combinations of deletion and missense variations of the dynein-2 DYNC2LI1 subunit found in skeletal ciliopathies cause ciliary defects

Cilia play crucial roles in sensing and transducing extracellular signals. Bidirectional protein trafficking within cilia is mediated by the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes, with the aid of kinesin-2 and dynein-2 motors. The dynein-2 complex drives retrograde trafficking of the IFT machinery after its transportation to the ciliary tip as an IFT cargo. Mutations in genes encoding the dynein-2-specific subunits (DYNC2H1, WDR60, WDR34, DYNC2LI1, and TCTEX1D2) are known to cause skeletal ciliopathies. We here demonstrate that several pathogenic variants of DYNC2LI1 are compromised regarding their ability to interact with DYNC2H1 and WDR60. When expressed in DYNC2LI1-knockout cells, deletion variants of DYNC2LI1 were unable to rescue the ciliary defects of these cells, whereas missense variants, as well as wild-type DYNC2LI1, restored the normal phenotype. DYNC2LI1-knockout cells coexpressing one pathogenic deletion variant together with wild-type DYNC2LI1 demonstrated a normal phenotype. In striking contrast, DYNC2LI1-knockout cells coexpressing the deletion variant in combination with a missense variant, which mimics the situation of cells of compound heterozygous ciliopathy individuals, demonstrated ciliary defects. Thus, DYNC2LI1 deletion variants found in individuals with skeletal ciliopathies cause ciliary defects when combined with a missense variant, which expressed on its own does not cause substantial defects.

The Significance of the DUF283 Domain for the Activity of Human Ribonuclease Dicer

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.

High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a “hot-spot”-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the “hot spot” of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

Sequential recognition of discrete export signals in flagellar subunits during bacterial Type III secretion

The flagellar T3SS delivers proteins from the bacterial cytosol to nascent cell surface flagella. Early subunits of the flagellar rod and hook are unchaperoned and contain their own export signals. One export signal, the gate recognition motif (GRM) docks subunits at the export gate, which must then open for unfolded subunits to enter the flagellar channel. Here, we identify a second signal at the extreme N-terminus of flagellar rod/hook subunits and determine that key to the signal is its hydrophobicity. We show that the two export signals are recognised sequentially, with the N-terminal signal being recognised only after subunits have docked at the export gate. The position of the N-terminal signal relative to the GRM is important, as a FlgD deletion variant (FlgDshort), in which the distance between the N-terminal signal and the GRM was shortened, stalled at the export machinery and was not exported. The attenuation of motility caused by FlgDshort was suppressed by mutations that destabilised the closed conformation of the FlhAB-FliPQR flagellar export gate, suggesting that the hydrophobic N-terminal signal might trigger gate opening.