[
Science,
2007]
In C. elegans, an effective RNA interference (RNAi) response requires the production of secondary siRNAs by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22 nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non-RISC (RNA-induced silencing complex) cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch, but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5'' di- or triphosphates and are only in minority associated with RDE-1, the RNAi-specific argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product.
[
Neuropathology,
2007]
Lewy bodies (LBs) are hallmark lesions in the brains of patients with Parkinson''s disease (PD) and dementia with Lewy bodies (DLB). We raised a monoclonal antibody LB509 against purified LBs from the brains of patients with DLB that strongly immunolabled LBs, and found that alpha-synuclein is one of the major components of LBs. Thus, the deposition of alpha-synuclein, an abundant presynaptic brain protein, as fibrillary aggregates in affected neurons or glial cells, was highlighted as a hallmark lesion of a subset of neurodegenerative disorders, including PD, DLB and multiple system atrophy collectively referred to as synucleinopathies. Importantly, the identification of missense mutations in and multiplication of alpha-synuclein gene in some pedigrees of familial PD has strongly implicated alpha-synuclein in the pathogenesis of PD and other synucleinopathies. We then examined the specific posttranslational modifications that characterize and underlie the aggregation of alpha-synuclein in synucleinopathy brains by mass spectrometry and using a specific antibody, and found that serine 129 of alpha-synuclein deposited in synucleinopathy lesions is selectively and extensively phosphorylated. Furthermore we generated transgenic C. elegans overexpressing alpha-synuclein in neurons, and found that overexpression of familial PD-linked mutant form of alpha-synuclein impairs functions of dopamine neurons. These findings collectively underscore the importance of deposition of alpha-synuclein as well as its phosphorylation in the pathogenesis of alpha-synucleinopathies.