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The endogenous ALF gene was characterized by RACE and RT-PCR for transcription start site usage, and by sodium bisulfite sequencing to determine its methylation status in somatic and oocyte tissues.
In the meantime, next-generation sequencing (NGS) technologies rapidly advanced, and as a consequence, experimental evidence for particular PA site usage can readily be generated by 3′ end sequencing.
Prior work suggests that increased CFIm25 dosage favors distal APA site usage for many transcripts, prominently including those for MECP2.
Similar to Mcl1, the predominant change was an increase in exon skipping (for BAX, GSK3B, HUWE1 and BTRC), while there was increased alternate splice donor site usage for BCL2L1 to generate Bclx-S preferentially over Bclx-L.
Transcription start site usage for the endogenous ALF gene involved primers S0 (5'-GATCATGGCCCATTCGGCTAACC-3'), S1 (5'-TGAACATTCATCAGCAACTTGG-3'), S2 (5'-CCCCTGACCATCAATAAAACAC-3'), S3 (5'-TGCGCAGACATGAGCCAGCGGA-3'), S4 (5'-GCAGCAGCGCGACGCGCAAAAG-3'), S5 (5'-GTCAGACCGCAGGCGATTGAAC-3'), and S6 (5'-TGGTTCCAACTTCCGGTTAGGC-3').
(B – E ) Shown is the relative change of the cleavage site usage for cleavage site 1 and cleavage site 2 based on in silico simulations of the polypeptide model with randomly sampled parameters.
Hence, we further developed a metric to characterize the overall splice site usage for every exon intron boundary based on an expression-weighted average of its inclusion ratio in all transcripts (Materials and Methods).
These 203 candidates showed evidence for differential promoter usage, polyadenylation site usage, ASE and whole gene expression changes.
To identify genes that employ the CoTC termination pathway we selected pre-mRNAs with a CstF-64 CLIP region in 3′ UTRs, extended by 200 bp downstream (to account for variability in poly(A) site usage and imprecision in transcript-end annotation).
