Figure 4.

Differential Processing of vtRNA1.1 into svRNAs in the Absence of m⁵C

(A) Schematics of secondary structure of vtRNA1.1 and small vault RNA (svRNA) found to be differentially abundant in NSUN2^+/− and NSUN2^−/− fibroblasts. CH3, cytosine-5 methylated site at position 69.

(B) Fold-change (log₂) and false discovery rate (FDR) values for reads of svRNA1-4 in NSUN2^+/− versus NSUN2^−/− human fibroblasts.

(C) Detection of svRNA1 and svRNA4 in NSUN2^+/− and NSUN2^−/− cells using qPCR.

(D and E) RNA levels of NSun2 (D) and svRNA4 (E) in NSun2 null (−/−) fibroblasts rescued by viral infection of NSun2 (pB-NSun2) compared to the empty vector control (pB-empty).

(F) Abundance of svRNA4 in NSUN2^−/− cell lysates (no-RNA) or incubated with synthetic vtRNA1.1 carrying (m⁵C) or lacking (no-m⁵C) at position 69.

(G) Detection of svRNA4 in small RNA pool copurified with Argonaute 2 (left) and Argonaute 3 (right). Let7-a and mir-150 are negative and mir-21 and mir-92-b are positive controls for Argonaute 2- and 3-bound microRNAs, respectively.

(H) qPCR showing expression of CACNG7 and CACNG8 RNA relative to GAPDH in NSUN2^−/− and NSUN2^+/− fibroblasts.

(I) Fold-change expression of CACNG7 and CACNG8 RNA in NSUN2^−/− and NSUN2^+/− fibroblasts transduced with svRNA antagomirs (as-svRNA4) or svRNA4 microRNA mimics (svRNA) versus respective control RNAs (ctr-RNA).

(J) RNA levels of CACNG7 and CACNG8 relative to GAPDH in NSun2 null (−/−) fibroblasts rescued by viral infection of NSun2 (pB-NSun2) compared to the empty vector control (pB-empty). Error estimates represent SEM (C–J).

See also Figures S5 and S6.