Figure 4

UM1-3 transcript start and terminator analysis. (A) 5'RACE analysis of the UM1-3 reporter gene transcripts (CeN7::GFP, CeN37::GFP and CeN74-2::GFP). Boxed bands correspond to the 5'RACE products of each reporter. The transcription start sites indicated by the 5'RACE bands of reporter constructs CeN7::GFP (UM1) and CeN74-2::GFP (UM3) are identical to the 5'ends of the mature endogenous CeN7 and CeN74-2 trancripts, respectively. The 5'RACE band from the CeN37::GFP (UM2) reporter constructed corresponds to a transcript starting 70 bp upstream of the 5'end of the mature endogenous CeN37 transcript (The lower band in the same lane is an unspecific band). (B) Distribution of C. elegans UM2 and UM3 ncRNAs terminator lengths. Frequency distribution analysis of poly(dT) terminator length was conducted on UM2 and UM3 ncRNA loci possessing a single RNA polymerase III termination signal starting within the first 40 bp downstream of the genomic sequence corresponding to the mature ncRNA. Inserted figures show data based on the analysis in ref [14]. (C) 3' RACE of CeN74-2_300 (UM3) and CeN37_1k (UM2). Bands corresponding to transcripts terminated at the 1^st, 2^nd and 3^rd "TTTT" run are indicated. (Sequence analysis suggests that the band between the second and third T run corresponds to transcript terminated at a T rich tract "TTCTTGTT"). (D) Relative expression level of transcripts terminated at the 1^st, 2^nd and 3^rd T run. qRT-PCR was performed by reverse transcription using the primer located in front of the 1^st, 2^nd and 3^rd T run, respectively, followed by a nested PCR.(see Methods for details).