ZCCHC3 protein expressed in 293T cells forms multimers and associates with L1 RNP complexes.

(A) ZCCHC3-FL and V5-TEV-ZCCHC3 proteins co-IP on α-FLAG-M2 affinity gel indicating their binding and multimerization. This is further supported by a higher molecular weight band visible at about 95 kD (top panel). In the lower panel we assume bands smaller than the 44 kD full-length ZCCHC3-FL are degradation products occurring during the IP and elution protocol. Vector only control was pcDNA6 myc-his B (pcDNA6). α-DYKDDDDK is an antibody product of Cell Signaling Technology and recognizes FLAG epitope tag. (B) FLAG-tagged ZCCHC3 IPed from HEK 293T cell lysates binds untagged L1 ORF1p complexes coexpressed from a full-length L1 (construct pc-L1-RP). ORF1p was detected by monoclonal α-ORF1-4H1 antibody [38]. Interaction was lost following treatment of lysates with RNase (+, last lane). (C) FLAG-HA-tagged ORF1p expressed from a full-length L1 (pc-L1-1FH) co-IPs V5-TEV-ZCCHC3 complexes from 293T cells. Interaction was diminished by treatment with RNase (+, last lane). (D) FLAG-tagged ZAP, MOV10, and ZCCHC3 co-IP with endogenous L1 ORF1p following affinity purification with α-FLAG-M2 antibody bound to Protein G Dynabeads (left). For this Western we used polyclonal α-L1Hs-ORF1p antibody. The bar chart (right) shows fold-enrichment of bound ORF1p after bands were quantified with ImageJ software (IP/ Input compared with empty vector). Association of ORF1p with the zinc-finger mutant ZCCHC3-FL-7AA was reduced 2-fold compared with wild-type ZCCHC3-FL. Empty vector pcDNA6 was used in duplicate as a negative control. (E) ZAP-L-FL, FL-MOV10, ZCCHC3-FL, and ZCCHC3-FL-7AA co-IP endogenous L1 RNA following affinity purification with α-FLAG-M2 antibody bound to Protein G Dynabeads. RT-qPCR was used to quantitate in the input and IP fractions L1 mRNA (blue bars; detected with the N51 primer pair targeting ORF1 sequence), and as controls β-actin mRNA (red bars) and GAPDH mRNA (green bars). Analyses by real-time RT-qPCR used the Relative Standard Curve Method. For each construct, we calculated the ratio of RNA of the IP fraction/ RNA of the input fraction compared with the empty vector (shown as fold enrichment on the y-axis). Results shown are the average of triplicate RT-qPCR reactions for one IP experiment. (F) α-ZCCHC3-CS antibody bound to Protein G Dynabeads was used in two separate IP reactions [1 and 2] to show that endogenous ZCCHC3 protein binds endogenous L1 ORF1p. Interaction was lost with RNaseA treatment (+, lanes 6 and 7). Normal rabbit IgG linked to Dynabeads was used as negative control and did not bind ORF1p (last two lanes). n = number of biological replicates.