Discrete subcellular partitioning of human retrotransposon RNAs despite a common mechanism of genome insertion

Abstract

Despite the immense significance retrotransposons have had for genome evolution much about their biology is unknown, including the processes of forming their ribonucleoprotein (RNP) particles and transporting them about the cell. Suppression of retrotransposon expression, together with the presence of retrotransposon sequence within numerous mRNAs, makes tracking endogenous L1 RNP particles in cells problematic. We overcame these difficulties by assaying in living and fixed cells tagged-RNPs generated from constructs expressing retrotransposition-competent L1s. In this way, we demonstrate for the first time the subcellular colocalization of L1 RNA and proteins ORF1p and ORF2p, and show their targeting together to cytoplasmic foci. Foci are often associated with markers of cytoplasmic stress granules. Furthermore, mutation analyses reveal that ORF1p can direct L1 RNP distribution within the cell. We also assayed RNA localization of the non-autonomous retrotransposons Alu and SVA. Despite a requirement for the L1 integration machinery, each manifests unique features of subcellular RNA distribution. In nuclei Alu RNA forms small round foci partially associated with marker proteins for coiled bodies, suborganelles involved in the processing of non-coding RNAs. SVA RNA patterning is distinctive, being cytoplasmic but without prominent foci and concentrated in large nuclear aggregates that often ring nucleoli. Such variability predicts significant differences in the life cycles of these elements.

The RIDL hypothesis: transposable elements as functional domains of long noncoding RNAs

“…In addition to being activity transcribed in cell compartment (Goodier et al 2010), developmental (Rowe and Trono 2011), and tissue-specific patterns (Faulkner et al 2009), many TE insertions are under purifying evolutionary selection (Lowe et al 2007). There is evidence for biological activity of repeats from a range of classes, from the large, autonomous long interspersed nuclear elements (LINEs), through various virally derived long terminal repeat (LTR) sequences, and to the nonautonomous short interspersed nuclear elements (SINEs).…”

L1 expression and regulation in humans and rodents

“…Both ORF1p and ORF2p localize predominantly to cytoplasm when overexpressed in human cell lines. In addition, the colocalization of L1 RNA and proteins is often associated with markers of cytoplasmic stress granules (149)(150)(151). Stress granules represent sites of stalled translation initiation complexes that accumulate in response to a variety of stress conditions (152).…”

“…Stress granules represent sites of stalled translation initiation complexes that accumulate in response to a variety of stress conditions (152). Thus, it is possible that cells recognize overexpression of L1 as a stressor and consequently sequester L1 RNP in stress granules for degradation (150). However, it remains unknown whether transit through stress granules by an L1 RNP is a requisite step for productive L1 retrotransposition (150, 151).…”

Stress out the LINEs

“…Goodier et al first reported the localization of LINE-1 RNP components with stress granule markers. 38 [24][25][26] co-localize with LINE-1 RNP in stress granules or P-bodies (Fig. 1).…”

“…37 Goodier et al have observed the localization of LINE-1 RNP to stress granules, albeit that the effect on LINE-1 activity was not fully investigated. 38,39 We have performed immunofluorescence experiments and utilized the automated Image Stream System to demonstrate that SAMHD1 expression causes sequestration of LINE-1 ORF1p in large cytoplasmic granule structures that are positively stained with stress granule markers G3BP1 and TIA1. This colocalization of LINE-1 components with stress granules was further confirmed by the results of co-immunoprecipitation and RT-PCR assays showing the association of stress granule marker protein G3BP1 with LINE-1 ORF1p and LINE-1 RNA.…”