# Role of the U-12 dependent Minor Spliceosome in Early Embryo Development and Brain Disease

> **NIH NIH F31** · WEILL MEDICAL COLL OF CORNELL UNIV · 2021 · $46,036

## Abstract

ABSTRACT
Alternative splicing is an imperative process that contributes to cellular specialization and systems complexity of
higher organisms. In mammals, alternative splicing is controlled by two macromolecular complexes: the major
(U2-dependent) and minor (U12-dependent) spliceosomes. While the U2-mediated spliceosome has been
extensively investigated, the U12-mediated spliceosome remains little understood. Recent studies in mice
indicate that loss of one minor spliceosome component causes early embryonic lethality. Although complete
absence of any minor spliceosome units has yet to be observed in humans, there are 9 disease phenotypes
associated with minor spliceosome dysfunction. Our lab was the first to report that a C84T nucleotide switch in
RNU12 causes human Early-Onset Cerebellar Ataxia (EOCA) and developmental delay. RNU12 encodes the
uridine-rich U12 small nuclear RNA (snRNA), which initiates minor spliceosome function through intron
recognition in pre-mRNAs. Leukocytes from homozygous RNU12C84T/C84T patients exhibited aberrant expression
of ataxia-related minor intron-containing genes (MIGs) and elevated intron retention, implicating an association
between deficient minor intron splicing and EOCA. Our preliminary data suggests that the RNU12 C84T variant
impairs U12-mediated splicing of cerebellar-specific pre-mRNAs, while complete RNU12 absence causes
early embryonic lethality due to aberrant splicing of key early developmental genes. This hypothesis will
be tested using next generation sequencing techniques, transcriptomics, embryo and brain development studies
in novel gene-edited mouse models, which contain either the C84T variant in mRnu12 (mRnu1284T), or a 79bp
deletion inactivating U12 snRNA (mRnu12—). AIM 1 will assess the role of the C84T RNU12 mutation in selective
mis-splicing of cerebellar transcripts through bulk RNA-sequencing of the cerebellum, contrasted with cortex and
hippocampus of mRnu1284T/84T mice and controls at different developmental timepoints. Using transcriptomic
tools, we will evaluate changes in gene expression, isoform usage, minor intron retention and splice site shift.
Candidate transcripts will be validated across genotypes, tissues and development using qRT-PCR and in situ
hybridization methods. AIM 2 will determine the impact of total RNU12 loss on embryo survival through lethality
studies of embryos from mRnu12+/— x mRnu12+/— mating pairs. Embryos will be examined and genotyped across
developmental stages to evaluate when mRnu12—/— embryos die. At a stage prior to mRnu12—/— loss, we will
analyze differences in MIG expression and splicing between mRnu12— mutants and controls using SMART-Seq2
technology and transcriptome analyses. Expression patterns of candidate MIG transcripts will be validated using
RT-PCR and immunohistochemical staining of embryo tissues. Together, these Aims will illuminate the critical
role of U12-mediated spliceosome function in mammalian development and its relevance to dise...

## Key facts

- **NIH application ID:** 10234959
- **Project number:** 1F31NS122531-01
- **Recipient organization:** WEILL MEDICAL COLL OF CORNELL UNIV
- **Principal Investigator:** Taylor Floyd
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $46,036
- **Award type:** 1
- **Project period:** 2021-09-30 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10234959

## Citation

> US National Institutes of Health, RePORTER application 10234959, Role of the U-12 dependent Minor Spliceosome in Early Embryo Development and Brain Disease (1F31NS122531-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10234959. Licensed CC0.

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