The LINC00961 transcript and its encoded micropeptide, small regulatory polypeptide of amino acid response, regulate endothelial cell function

Abstract

Aims Long non-coding RNAs (lncRNAs) play functional roles in physiology and disease, yet understanding of their contribution to endothelial cell (EC) function is incomplete. We identified lncRNAs regulated during EC differentiation and investigated the role of LINC00961 and its encoded micropeptide, small regulatory polypeptide of amino acid response (SPAAR), in EC function. Methods and results Deep sequencing of human embryonic stem cell differentiation to ECs was combined with Encyclopedia of DNA Elements (ENCODE) RNA-seq data from vascular cells, identifying 278 endothelial enriched genes, including 6 lncRNAs. Expression of LINC00961, first annotated as an lncRNA but reassigned as a protein-coding gene for the SPAAR micropeptide, was increased during the differentiation and was EC enriched. LINC00961 transcript depletion significantly reduced EC adhesion, tube formation, migration, proliferation, and barrier integrity in primary ECs. Overexpression of the SPAAR open reading frame increased tubule formation; however, overexpression of the full-length transcript did not, despite production of SPAAR. Furthermore, overexpression of an ATG mutant of the full-length transcript reduced network formation, suggesting a bona fide non-coding RNA function of the transcript with opposing effects to SPAAR. As the LINC00961 locus is conserved in mouse, we generated an LINC00961 locus knockout (KO) mouse that underwent hind limb ischaemia (HLI) to investigate the angiogenic role of this locus in vivo. In agreement with in vitro data, KO animals had a reduced capillary density in the ischaemic adductor muscle after 7 days. Finally, to characterize LINC00961 and SPAAR independent functions in ECs, we performed pull-downs of both molecules and identified protein-binding partners. LINC00961 RNA binds the G-actin sequestering protein thymosin beta-4x (Tβ4) and Tβ4 depletion phenocopied the overexpression of the ATG mutant. SPAAR binding partners included the actin-binding protein, SYNE1. Conclusion The LINC00961 locus regulates EC function in vitro and in vivo. The gene produces two molecules with opposing effects on angiogenesis: SPAAR and LINC00961.

The Influence of the LINC00961/SPAAR Locus Loss on Murine Development, Myocardial Dynamics, and Cardiac Response to Myocardial Infarction

“…We have previously reported that LINC00961 locus CRISPR/Cas9 KO reduces alpha-smooth muscle actin (αSMA) cell-density in the mouse hindlimb skeletal muscle without altering capillary density. [16] Global dysfunction in EC endowment resulting in compromised development of the vascular network could limit the capacity for myocardial growth and the maintenance of functional myocardium throughout life. [20] Reduced LV volume independent of adult size in LINC00961 -/-mice suggests that the vascular influence on cardiac development requires further investigation.…”

“…Cells were lysed with QIAzol (QIAGEN, USA), RNA extracted and qRT-PCR run as previously described. [6,16] 4…”

“…Additionally, we have previously demonstrated that the LINC00961 locus directly regulates EC function, with SPAAR and LINC00961 counter-regulating angiogenesis in vitro and evidenced in a murine hindlimb ischaemia model of critical limb ischaemia. [16] In our previous work, we showed that LINC00961 binds the actinbinding protein thymosin beta-4x, which is involved in regulating cytoskeletal dynamics, angiogenesis and cell migration, [17] while SPAAR binds the actin-binding protein spectrin repeat containing nuclear envelope protein 1 (SYNE1/NESPRIN-1), which is highly expressed in cardiomyocytes. [18] These initial data suggest the importance of the LINC00961/SPAAR locus in cellular maintenance and response to environmental insult.…”

Evaluation of the potential role of long non-coding RNA LINC00961 in luminal breast cancer: a case–control and systems biology study

“…According to the role of LINC00961 in different cancers that has been proved in previous studies, LINC00961 could play crucial roles in tumor suppression in various cancers. It has recently been determined that LINC00961 encodes a conserved polypeptide, named small regulatory polypeptide of amino acid response (SPAAR) which modulates skeletal muscle regeneration [15]. Although, the expression pattern and biological function of LINC00961 have been studied in many human cancers, to the best of our knowledge, the present study is the first project that investigates the role of this lncRNA in luminal BC so far.…”

LncRNAs interacting with the translation machinery contribute to human neuronal differentiation

“…Predicting which lncRNA-smORFs are translated has proven difficult, as attributes to predict protein-coding ability are ineffective on small non-canonical ORFs. A small but growing number of smORF peptides translated from lncRNAs have been found to exhibit cellular and organismal functions (Anderson et al, 2015;Chen et al, 2020;Magny et al, 2013;Pueyo & Couso, 2008;Spencer et al, 2020;Wang et al, 2020).…”