Investigating RNA metabolism during neuronal development in human models for Prader-Willi syndrome

Abstract

The human brain expresses numerous RNA molecules and RNA-associated proteins, many of which remain poorly characterised despite their potential importance in neuronal development and function. One such RNA class are small nucleolar RNAs (snoRNAs) that associate with proteins to form small nucleolar ribonucleoproteins (snoRNPs) to typically guide ribosomal RNA (rRNA) modifications. Prader–Willi syndrome (PWS) is a neurodevelopmental disorder characterised by delayed cognitive and sexual development, hyperphagia-induced obesity, and hypogonadism. In 65–75% of cases, PWS arises from deletions in the paternal 15q11.2–q13 region, which contains neuronal-specific box C/D snoRNAs SNORD115 and SNORD116, each present in multiple tandem copies. The maternal allele is silenced by imprinting. Deletion sizes vary, with the smallest deletion of approximately 100–200 kb primarily removing SNORD116. While the targets and mechanisms of these snoRNAs remain unknown, their accumulation during neuronal differentiation suggests a role in later neurodevelopment.

Small RNA sequencing (RNA-seq) of heterozygous deletion mutant cell lines lacking either SNORD115 or SNORD116 expression investigated how these two neuronal-specific snoRNAs alter the expression of other non-coding RNAs (ncRNAs) during neurodevelopment. This identified numerous snoRNAs with dysregulated expression changes during neuronal differentiation. The multiple tandem repeats in the SNORD115 and SNORD116 clusters are related, but non-identical, and differences in relative accumulation were also observed.

The role of non-essential Fibrillarin-like 1 (FBLL1) was tested to investigate what features result in snoRNA accumulation during neurodevelopment. FBLL1 is a largely uncharacterised, but 83% identical paralog of the essential protein Fibrillarin (FBL). Box C/D snoRNAs, which include both SNORD115 and SNORD116, associate with four common proteins: FBL, NHP2L1, NOP56, and NOP58. FBLL1 is proposed to functionally replace FBL in neuronal cells and to test this, it was confirmed that cells with CRISPR-mediated deletions in the 5’ region of FBLL1 did not have significantly altered expression of any snoRNAs. However, there were other significant transcriptomic and proteomic effects resulting from FBLL1 gene knockout, many of which are associated with normal neuronal function. Analysis of protein–protein interactions confirmed that FBLL1 assembles into the canonical box C/D snoRNP complex similar to FBL, enabling the same RNA 2′-O-methylation function, while also identifying novel interaction partners. Both FBL and FBLL1 proteins also localise to regions within the nucleus of neuronal cells.

Investigating snoRNA targets and mechanisms in PWS, including the function of FBLL1, elucidates the roles of neuronal snoRNAs and advances understanding of neuronal cell biology. Such studies may reveal molecular mechanisms underlying PWS and other RNA-linked cognitive disorders, providing potential avenues for targeted interventions.