Extended Data Fig. 8: ZCCHC7 protein expression in lymphoma tissue, ZCCHC7 interaction network, reconstituted structure of the TRAMP complex using alpha-fold, and ZCCHC7 localization in SUDHL6 cells.
(a) Tissue microarrays (TMAs) were immunohistochemically stained for ZCCHC7. DLBCL (N = 33, p = 0.0084) and DHL (N = 9, p = 0.03) showed a higher average degree of ZCCHC7 expression than individual benign lymph node, spleen, or tonsil, all of which showed a very low degree of ZCCHC7 expression. Mean H-score and standard deviation for each group are shown, with p-values calculated by two-sided Wilcoxon rank-sum test. (b) Longitudinal samples representing transformation of follicular lymphoma to DLBCL in 10 patients (separate from the sequenced cohort) were stained for ZCCHC7. ZCCHC7 expression usually increased upon lymphoma transformation. (c) Comparison of endogenous ZCCHC7 in the non-neoplastic human B cell line CL-01 and SUDHL6 (a DHL line that overexpresses ZCCHC7). NPM1 defines the nucleolus and DAPI counterstains nuclei (blue). ZCCHC7 accumulates in nucleoli of CL01 cells while also present in the nucleoplasm of SUDHL6 (scale bar 5 µm). (d) The ratio of nucleolar to total nuclear signal is significantly reduced in SUDHL6 compared to CL-01. Statistical significance was assessed using an unpaired two-tailed t-test, and the number of cells analyzed in each case is indicated. (e) Pre-rRNA processing pathway. Three out of four mature rRNAs, the 18S, 5.8S, and 28S are encoded as a long polycistronic precursor synthesized by RNA polymerase I, the 47S. The mature rRNA sequences are produced by extensive processing (cleavage sites indicated in blue). In the 47S, the mature rRNAs are interspersed by 5′ and 3′ external transcribed spacers (ETS) and internal transcribed spacers (ITS) 1 and 2. The probes used in northern blotting (LD1828, LD1844, LD2079, and LD2122) are highlighted. (f) FLAG-tagged ZCCHC7 protein and a FLAG-tag peptide were overexpressed in 293T cells to purify ZCCHC7 interacting factors via Mass spectrometry. The components of the RNA exosome complex essential cofactor NEXT directly interact with ZCCHC7, as does a set of nucleolar proteins. (g) Predicted structure model of the human TRAMP-like complex. The complex consists of the RNA helicase MTR4 (PDB ID: 7S7B, yellow), RNA (PDB ID: 7S7B, blue), the zinc-knuckle protein ZCCHC7 (AlphaFold prediction, green), and the noncanonical poly(A) polymerase PAPD5 (AlphaFold prediction, red). Ribbon representation of the model (left) and 90° rotation (right) highlighting binding of RNA and its recruitment to the RNA channel in MTR4.