Reduced levels of protein recoding by A-to-I RNA editing in Alzheimer's disease

Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer''s disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing.     

Temperature-dependent RNA editing in octopus extensively recodes the neural proteome

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On the origin and evolution of RNA editing in metazoans

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Positive charge in the K-loop of the kinesin-3 motor KIF1A regulates superprocessivity by enhancing microtubule affinity in the one-head–bound state

KIF1A is an essential neuronal transport motor protein in the kinesin-3 family, known for its superprocessive motility. However, structural features underlying this function are unclear. Here, we determined that superprocessivity of KIF1A dimers originates from a unique structural domain, the lysine-rich insertion in loop-12 termed the ‘K-loop’, which enhances electrostatic interactions between the motor and the microtubule. In 80 mM PIPES buffer, replacing the native KIF1A loop-12 with that of kinesin-1 resulted in a 6-fold decrease in run length, whereas adding additional positive charge to loop-12 enhanced the run length. Interestingly, swapping the KIF1A loop-12 into kinesin-1 did not enhance its run length, consistent with the two motor families using different mechanochemical tuning to achieve persistent transport. To investigate the mechanism by which the KIF1A K-loop enhances processivity, we used microtubule pelleting and single-molecule dwell time assays in ATP and ADP. First, the microtubule affinity was similar in ATP and in ADP, consistent with the motor spending the majority of its cycle in a weakly bound state. Second, the microtubule affinity and single-molecule dwell time in ADP were 6-fold lower in the loop-swap mutant than WT. Thus, the positive charge in loop-12 of KIF1A enhances the run length by stabilizing binding of the motor in its vulnerable one-head–bound state. Finally, through a series of mutants with varying positive charge in the K-loop, we found that KIF1A processivity is linearly dependent on the charge of loop-12, further highlighting how loop-12 contributes to the function of this key motor protein.     

In vivo roles of the basic domain of dynactin p150 in microtubule plus-end tracking and dynein function

Microtubule (MT) plus-end-tracking proteins accumulate at MT plus ends for various cellular functions, but their targeting mechanisms are not fully understood (Akhmanova A and Steinmetz MO. Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat Rev Mol Cell Biol 2008;9:309-322.). Here, we tested in the filamentous fungus Aspergillus nidulans the requirement for plus-end localization of dynactin p150, a protein essential for dynein function. Deletion of the N-terminal MT-binding region of p150 significantly diminishes the MT plus-end accumulation of both dynein heavy chain and p150, and causes a partial defect in nuclear distribution. Surprisingly, within the MT-binding region, the basic domain is more critical than the CAP-Gly (cytoskeleton-associated protein glycine-rich) domain for maintaining plus-end tracking of p150, as well as for the functions of dynein in nuclear distribution and early endosome movement. Our results show that the basic domain of A. nidulans p150 is important for p150-MT interaction both in vivo and in vitro, and the basic amino acids within this domain are crucial for the plus-end accumulation of p150 in the wild-type background and for the p150-MT interaction in the ΔkinA (kinesin-1) background. We suggest that the basic amino acids are required for the electrostatic interaction between p150 and MTs, which is important for kinesin-1-mediated plus-end targeting of dynactin and dynein in A. nidulans.     

The domain of unknown function 4005 (DUF4005) in an Arabidopsis IQD protein functions in microtubule binding

The dynamic responses of microtubules (MTs) to internal and external signals are modulated by a plethora of microtubule-associated proteins (MAPs). In higher plants, many plant-specific MAPs have emerged during evolution as advantageous to their sessile lifestyle. Some members of the IQ67 domain (IQD) protein family have been shown to be plant-specific MAPs. However, the mechanisms of interaction between IQD proteins and MTs remain elusive. Here we demonstrate that the domain of unknown function 4005 (DUF4005) of the Arabidopsis IQD family protein ABS6/AtIQD16 is a novel MT-binding domain. Cosedimentation assays showed that the DUF4005 domain binds directly to MTs in vitro. GFP-labeled DUF4005 also decorates all types of MT arrays tested in vivo. Furthermore, we showed that a conserved stretch of 15 amino acid residues within the DUF4005 domain, which shares sequence similarity with the C-terminal MT-binding domain of human MAP Kif18A, is required for the binding to MTs. Transgenic lines overexpressing the DUF4005 domain displayed a spectrum of developmental defects, including spiral growth and stunted growth at the organismal level. At the cellular level, DUF4005 overexpression caused defects in epidermal pavement cell and trichome morphogenesis, as well as abnormal anisotropic cell elongation in the hypocotyls of dark-grown seedlings. These data establish that the DUF4005 domain of ABS6/AtIQD16 is a new MT-binding domain, overexpression of which perturbs MT homeostasis in plants. Our findings provide new insights into the MT-binding mechanisms of plant IQD proteins.     

In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.