RNA-seq data analysis has revealed abundant alternative splicing in eukaryotic mRNAs. However, splicing is only one of many processing events that transcripts may undergo during their lifetime. We present here RNAprof (RNA profile analysis), a program for the detection of differential processing events from the comparison of RNA-seq experiments. RNAprof implements a specific gene-level normalization procedure and compares RNA-seq coverage profiles at nucleotide resolution to detect regions of significant coverage differences, independently of splice sites or other gene features. We used RNAprof to analyze the effect of alternative-splicing regulators NSRa and NSRb on the Arabidopsis thaliana transcriptome. A number of intron retention events and alternative transcript structures were specifically detected by RNAprof and confirmed by qRT-PCR. Further tests using a public Mus musculus RNA-seq dataset and comparisons with other RNA isoform predictors showed that RNAprof uniquely identified sets of highly significant processing events as well as other relevant library-specific differences in RNA-seq profiles. This highlights an important layer of variation that remains undetected by current protocols for RNA-seq analysis.