Figure 2.

Rdp1 is required neither to generate TERRA nor C-rich telomeric RNA. (A) RT-PCR reveals telomeric and subtelomeric RNA transcribed from the subtelomere outwards. Top: Schematic of telomeric region. Oligonucleotides used are designated as arrows pointing in the 5′-to-3′ direction. First strand synthesis with or without reverse transcriptase (RT+/−) using the primers indicated (first strand) was performed on RNA from the indicated strains. cDNA was then amplified with the oligonucleotides indicated (PCR). Genomic DNA (DNA ctrl) used as template was included as a control. o3 primes cDNA from RNA transcribed in a telomere-to-subtelomere direction, whereas oC primes cDNA from RNA of the opposite polarity. oG likely primes C-rich telomeric cDNA, but subtelomeric primers would not amplify this cDNA. This serves as an important negative control, as under standard denaturation conditions, endogenous priming of telomeric RNA can occur; such endogenous priming was removed under the conditions used here (see ‘Materials and Methods’ section). The act1 control demonstrates the presence of RNA in all samples. (B) Transcription of the telomeric C-strand is independent of Rdp1. Strand-specific northern blot analysis of total cellular RNA as in Figure 1D. Telomere-specific signals are absent from the blot probed with C-rich RNA, as all signals are present in the Otrt1Δ sample. In contrast, the G-rich probe detects telomere-specific C-strand signal in taz1Δ and taz1Δrdp1Δ samples. C-rich and G-rich probes hybridize approximately equally to ds telomeric DNA (data not shown).