Tyro3 Modulates Mertk-Associated Retinal Degeneration.

Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.

Interaction of host cell microRNAs with the HCV RNA genome during infection of liver cells.

It has remained an enigma how hepatitis C viral (HCV) RNA can persist in the liver of infected patients for many decades. With the recent discovery of roles for microRNAs in gene expression, it was reported that the HCV RNA genome subverts liver-specific microRNA miR-122 to protect its 5' end from degradation by host cell exoribonucleases. Sequestration of miR-122 in cultured liver cells and in the liver of chimpanzees by small, modified antisense RNAs resulted in dramatic loss of HCV RNA and viral yield. This finding led to the first successful human trial in which subcutaneous administration of antisense molecules against miR-122 lowered viral yield in HCV patients, without the emergence of resistant virus. In this review, the authors summarize the molecular mechanism by which miR-122 protects the HCV RNA genome from degradation by exoribonucleases Xrn1 and Xrn2 and discuss the application of miR-122 antisense molecules in the clinic.

Hepatitis C virus subverts liver-specific miR-122 to protect the viral genome from exoribonuclease Xrn2.

The abundant, liver-specific microRNA miR-122 forms extensive base-pairing interactions with the 5' noncoding region of the hepatitis C virus (HCV) RNA genome, protecting the viral RNA from degradation. We discovered that the 5'-3' exoribonuclease Xrn2, which plays a crucial role in the transcription termination of RNA polymerase II, modulates HCV RNA abundance in the cytoplasm, but is counteracted by miR-122-mediated protection. Specifically, Xrn2 depletion results in increased accumulation of viral RNA, while Xrn2 overexpression diminishes viral RNA abundance. Depletion of Xrn2 did not alter translation or replication rates of HCV RNA, but affected viral RNA stability. Importantly, during sequestration of miR-122, Xrn2 depletion restored HCV RNA abundance, arguing that Xrn2 depletion eliminates the miR-122 requirement for viral RNA stability. Thus, Xrn2 has a cytoplasmic, antiviral function against HCV that is counteracted by HCV's subversion of miR-122 to form a protective oligomeric complex at the 5' end of the viral genome.

Aptamer-based endocytosis of a lysosomal enzyme.

Enzyme replacement therapy for lysosomal storage diseases is currently based on endocytosis of lysosomal enzymes via the mannose or mannose 6-phosphate receptors. We are developing a technology for endocytosis of lysosomal enzymes that depends on generic, chemically conjugated reagents. These reagents are aptamers (single-stranded nucleic acid molecules) selected to bind to the extracellular domain of the mouse transferrin receptor. After selection, an RNA aptamer and a DNA aptamer were modified with biotin and linked to dye-labeled streptavidin for detection by confocal microscopy. Aptamer-streptavidin conjugates showed saturable uptake into mouse fibroblasts (Ltk(-) cells), which could be inhibited by an excess of free aptamer but not by tRNA, calf thymus DNA, or transferrin. The RNA aptamer-streptavidin conjugate was mouse-specific, as human cells (293T) did not take it up unless first transfected with the mouse transferrin receptor. Some streptavidin separated from the recycling pathway of transferrin and colocalized with lysosomes. After characterization in the model system, the DNA aptamer was conjugated to a lysosomal enzyme, alpha-l-iduronidase, from which mannose 6-phosphate had been removed. The aptamer had been modified by attachment of terminal glycerol for oxidation by periodate and reaction of the resulting aldehyde with amino groups on the protein. Dephospho-alpha-L-iduronidase-aptamer conjugate was taken up in saturable manner by alpha-L-iduronidase-deficient mouse fibroblasts, with half-maximal uptake estimated as 1.6 nM. Endocytosed enzyme-aptamer conjugate corrected glycosaminoglycan accumulation, indicating that it reached lysosomes and was functional in those organelles. Both uptake and correction were inhibited by unconjugated aptamer, confirming the role of the aptamer in receptor-mediated endocytosis.