GASZ Is Essential for Male Meiosis Figure 5. GASZ interactions and reduction of nuage proteins in Gasz testes. Biosensor quantification of interaction for a full-length GASZ bait by (A) RANBP9 partial clone, and by (B) GASZ and MIWI. (C,D) GASZ co-immunoprecipitation with nuage proteins. Testicular protein lysates from 21-day-old mice were incubated with no primary antibody [lane 1, (C,D)], anti-GASZ antibody [lane 2, (C)] or anti-MIWI antibody [lane 2, (D)] to immunoprecipitate protein complexes. 10 mg of WT lysate [lane 3, (C,D)] and Gasz2/2 lysate [(lane 4, (C)] were used as controls. Co- immunoprecipitating proteins were detected by western blot analysis using antibodies against MIWI, TDRD1, MVH, MILI, MAEL, TDRD6, TDRD7, or b- actin. (E) Western blot analysis of testicular protein lysates prepared from 10- and 14-day-old Gasz+/2 (+/2, lanes 1 and 3) and Gasz2/2 (2/2, lanes 2 and 4) mice. Antibodies against MIWI, TDRD1, MVH, MILI, TDRD6, TDRD7, or an antibody to b-actin show levels of nuage proteins are reduced in Gasz2/2 testes prior to spermatocyte apoptosis. doi:10.1371/journal.pgen.1000635.g005 S2). Twenty-seven of the 100 small RNAs displaying a relative increase in Gasz2/2 testes appeared to be variants of miRNAs or potential passenger strands, many mapping to a miRNA cluster on the X chromosome (Table S3). Two additional 22–23 nt sequences, DQ712837 mapping to Small Cajal body specific RNA 15 (Scarna15) and DQ688886 mapping to an intron of 1700041C02Rik, contributed 40% of the ‘‘piRNA’’ reads remain- ing in Gasz2/2 testes. The 127 nt Scarna15 is predicted to form two stem-loops and DQ712837 derives from the stem of the second stem-loop. If we exclude these two likely misannotated Dicer- dependent small RNAs, the non-repeat-associated piRNAs reads are 99% reduced. Control testes express an abundance of repeat-associated small RNAs compared to the Gasz2/2 testes (Figure 7A and Table S4), which fall into two size categories that peak at 22 and 27 nt (Figure S10A). While there is a small peak at 22 nt in Gasz2/2 testes, the 27 nt peak is essentially absent. To analyze the repeat-associated piRNAs, we excluded all sequences whose length was not 25– 29 nt. Many of the 19–23 nt sequences map to SINEs (Figure S10D and Figure S10E and Table S3) and typically show less than a two-fold change. These SINE-associated small RNAs belong to a novel small RNA class that are cleaved from their precursor RNA by a Dicer-dependent but DGCR8-independent mechanism [53]. We also excluded 19–23 nt sequences that did not map to SINEs as potential Dicer-dependent endo-siRNAs (Figure S10F and Figure S10G). All 25–29 nt repeat-associated RNAs were classified as repeat-associated piRNAs (Figure 7C). These repeat-associated piRNAs map to LTRs (e.g., ERV-K IAPLTR1a_I_MM and MaLR MTA_Mm_LTR) or LINEs (L1_MM), and are 10 to 100- fold less abundant in Gasz2/2 testes (Figure S10B and Figure S10C and Table S4). The majority of the 25–29 nt repeat- associated piRNAs contained a U at position 1 and an A at position 10 (Figure S10H), characteristic of participation in the ‘‘ping-pong’’ synthesis reaction [54]. In contrast, the 19–23 nt repeat-associated small RNAs had an initial U but a variable position 10 (Figure S10I), suggesting a lack of amplification by the ‘‘ping-pong’’ mechanism and confirming that these sequences are unlikely to be synthesized by the piRNA machinery. Subsequent analysis showed that the biggest difference in repeat-associated piRNAs occurred at P7 when many more elements were affected. Thus, the defect in repeat-associated piRNA production preceding delayed meiotic prophase initiation in Gasz2/2 testes is a cause rather than an effect of this process. Initially, 25% of the small RNAs sequences (.1 million reads) in control testes were unclassified. The majority of the unclassified sequences were 25–29 nt (Figure 7D and Figure S10J), but a 19– 23 nt class was also present (Figure S10K). The majority of these unclassified RNAs possess a 59 U but a variable 10th position (Figure S10L). Because of their similarity to piRNAs, we term the 25–29 nt category of unclassified sequences as ‘‘putative piRNAs’’ while the smaller size category called ‘‘unknown small RNAs’’ may contain novel miRNAs or other Dicer-dependent small RNAs. ‘‘Putative piRNA’’ reads were reduced compared to controls at P7 (7.9% versus 0.29%), P10 (1.6% versus 16.9%) and P14 (5.10% PLoS Genetics | www.plosgenetics.org 7 September 2009 | Volume 5 | Issue 9 | e1000635