Figure 1.
Y RNAs associate with processing factors and promote histone pre-mRNA processing. (A,B) RNA affinity purification with the indicated Y RNAs was performed in HEK293 cell lysates. Copurification of proteins (RBPs and processing factors) was determined by Western blotting with the indicated antibodies. (C) Bead control; (I) input fraction (5% of total); (*) cross-reactivity of anti-SYMPK. (C) HEK293 cells were transfected with control (C) or the indicated ncRNA-directed antisense oligonucleotides (ASOs) for 48 h. The depletion of ncRNAs was determined by infrared Northern blotting of total RNA with the indicated probes. 5S and Y5 ncRNAs served as loading controls. (D) Schematic of nonhistone and histone mRNAs, including the coding sequence (CDS) and the cleavage site (arrow), following the cleavage signals (PAS: AAUAAA or SL structure). Colored double arrows indicate the PCR products used for the quantification of (1) total nonhistone mRNAs (T; yellow), (2) misprocessed nonhistone mRNAs (MP; red), (3) total histone mRNAs (T; green), and (4) misprocessed histone mRNAs (MP; blue). (E) HEK293 cells were transfected with the indicated ASOs as in C as well as CPSF1-directed siRNAs. The levels of total (T) and misprocessed (MP) mRNAs (ACTB, EEF2, H2AC: HIST1H2AC; and H3A: HIST2H3A) were analyzed by quantitative RT–PCR (qRT–PCR) using R6 priming as depicted in D. The fold change of transcript levels was determined by the ΔΔCT method relative to cells transfected with control ASOs/siRNAs using the PPIA-encoding mRNA for normalization. (F) HEK293 cells were transfected with control ASO (ASOC) or three distinct Y3-directed ASOs. The fold change of misprocessed H2AC and H3A mRNAs was determined as in E. The depletion of Y3 was monitored by Northern blotting as in C. Error bars indicate the SD of at least three independent analyses. Statistical significance was determined by Student's t-test, (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.