Characterization of the piRNA Complex from Rat Testes

Nelson C. Lau, Anita G. Seto, Jinkuk Kim, Satomi Kuramochi-Miyagawa, Toru Nakano, David P. Bartel, Robert E. Kingston

SCIENCE (313), 21 JULY 2006

Rat Testes contains longer class of small RNAs

Rat testes extract was fractionated on a Q column (0.1 to 1 M potassium acetate gradient). RNA from fractions was endlabeled and resolved on a gel. Small RNAs from column fractions were gel-purified (dashed boxes), converted to cDNAs, and sequenced.

These small RNAs belong to a unique class

These RNAs have tissue specific expression pattern

Size distribution of small RNAs from FT (white), and eluate (black).
Y-axis scales are different for flowthrough RNAs (left) and eluate RNAs (right).

Rat tissue Northern blot hybridized with body-labeled RNA probes corresponding to small RNA sequences.

Purification of native small RNA containing complex

Schematic of piRC fractionation steps from rat testes extract
Numbers represent potassium acetate (KOAc) concentration (mM).

Small RNAs were end-labeled and resolved on a gel Western blots probed with antibodies to Miwi (mouse Piwi) and to hRecQ1

Small RNA co-purifies with Riwi and rRecQ1

Insights into the origin of the piRNAs

Northern blot analysis with probes to the indicated clusters, testing strandspecific piRNA expression (left and middle) and cross-hybridization to mouse and human testes RNAs (right)

Biochemical Characterization of piRC

Fractions containing rRecQ1 exhibit ATPase activity

Visualization of proteins and endlabeled small RNAs in fractions from final Superdex-200 column

Biochemical Characterization of piRC

Fractions containing rRecQ1 exhibited DNA unwinding activity

Fractions containing Riwi and piRNAs exhibit slicer activity

rRecQ1 exhibits ATP dependant DNA helicase Riwi shows slicing activity