Human cells contain myriad excised linear intron RNAs with links to gene regulation and potential utility as biomarkers

Previous Thermostable Group II Intron Reverse Transcriptase sequencing (TGIRT-seq) found that human plasma contains short (≤300 nt) structured full-length excised linear intron RNAs (FLEXIs) with potential utility as blood-based biomarkers. Here, TGIRT-seq identified >9,000 different FLEXIs in human cells, including relatively abundant FLEXIs with cell-type-specific expression patterns. Analysis of published CLIP-seq datasets identified 126 RNA-binding proteins (RBPs) that bind different FLEXIs, including 53 RBPs that bind ≥30 FLEXIs. These RBPs included splicing factors, transcription factors, a chromatin remodeling protein, cellular growth regulators, and proteins with cytoplasmic functions. Analysis of published datasets identified subsets of these RBPs that bind at FLEXI splice sites and impact alternative splicing or FLEXI host gene mRNA levels. Hierarchical clustering identified 6 subsets of RBPs whose binding sites were co-enriched in different subsets of FLEXIs: AGO1-4 and DICER, including but not limited to annotated agotrons and mirtron pre-miRNAs; DKC1, NOLC1, SMNDC1, and AATF (Apoptosis Antagonizing Transcription Factor), including but not limited to FLEXIs encoding snoRNAs; two sets of alternative splicing factors; and two sets that included RBPs with cytoplasmic functions ( e.g. , LARP4, PABPC4, METAP2, and ZNF622) together with nuclear regulatory proteins. The subsets of host genes encoding FLEXIs that bind these RBPs were enriched with non-FLEXI other short and long introns that bind the same RBPs, and TGIRT-seq of nuclear and cytoplasmic fractions from 4 cell lines showed cytoplasmic enrichment of FLEXIs with binding sites for RBPs that function in the cytoplasm. Collectively, our findings suggest that different subsets of RBPs bind FLEXIs and other introns to coordinately regulate the expression of functionally related host genes and then co-localize with stably bound FLEXIs to different intracellular locations. The cell-type specific expression of relatively abundant FLEXIs suggests utility as cellular as well as blood-based RNA biomarkers. ### Competing Interest Statement AML is an inventor on patents owned by the University of Texas at Austin for TGIRT enzymes and other stabilized reverse transcriptase fusion proteins and methods for non-retroviral reverse transcriptase template switching. AML, JY, and HX are inventors on a patent application filed by the University of Texas for the use of FLEXIs and other intron RNAs as biomarkers.