Multivalent interactions between RNA and RNA binding proteins drive differential intra-speckle positioning of RNA transcripts

Nuclear speckles represent a type of membraneless organelles in higher eukaryotic cells which are rich in snRNP components, certain splicing factors, including SR proteins (a family of RNA binding proteins (RBPs) named for containing regions with repetitive serine and arginine residues), polyA+ RNAs, and certain long noncoding RNAs (lncRNA). Multivalent interactions between RNAs and residing protein components drive the formation, as well as layered organization of components in nuclear speckles. Furthermore, RBPs have differential localization with respect to nuclear speckles; while SRSF1 proteins is enriched in nuclear speckles, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is either uniformly distributed in the nucleoplasm or slightly depleted from nuclear speckles. Using super-resolution imaging we show that the pre-mRNA transcripts containing SRSF1 motifs in the exon and hnRNPA1 motifs in the intron, are orientated in a way such that the regions containing the SRSF1 binding motifs are positioned relatively towards the interior of nuclear speckles and hnRNPA1 binding motifs are placed relatively towards the periphery of speckles. Knocking down of SRSF1 protein leads to the migration of RNA transcripts further towards the periphery of nuclear speckles, whereas knocking down of hnRNPA1 protein leads to the inward migration of RNA transcripts with respect to the speckle center. A similar effect of protein knockdown is seen on the endogenous nuclear speckle associated lncRNA, MALAT1. Our results collectively demonstrate that RNAs can adopt non-random orientation and positioning within the membraneless organelles. These observations might help explain the need for nuclear RNAs for structural integrity of nuclear speckles and the context dependent effect of SR and hnRNP proteins on splicing outcomes.