H2020Индивидуална стипендия2022–2023

TITINmap · Towards Uncovering the Splicing Code of the Gigantic Gene Titin in Familial Dilated Cardiomyopathy

„Хоризонт 2020“ — Действия „Мария Склодовска-Кюри“

Период: 2022-01-01 → 2023-12-31
Финансиране от ЕС: 176 036 €
Участници: 2
Схема: MSCA-IF

Линиите свързват координатора с партньорите.

Накратко на български

Протеинът титин и начинът, по който генът RBM20 контролира неговата структура, се анализират чрез редактиране на гени и секвениране на единични клетки. Това помага за разбирането на фамилната дилатирана кардиомиопатия и търсенето на стратегии за коригиране на дефектите при пациентите.

Този кратък обзор е генериран от изкуствен интелект

Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.

Резултати накратко

Towards Uncovering the Splicing Code of the Gigantic Gene Titin in Familial Dilated Cardiomyopathy

Titin (TTN) is the largest protein of the human body and critical for the contraction of heart muscle cells. TTN mutations are prevalent in patients with familial dilated cardiomyopathy (DCM), a common heart disease that is a global threat to the aging society. RBM20, which is also mutated in patients with an aggressive form of DCM, regulates the length and function of TTN by a process named alternative splicing. How RBM20 mediates alternative splicing of TTN is largely unknown. My fellowship project was to develop new methods to analyze RBM20-dependent splicing of TTN and other crucial targets in single cells. To this end, I performed CRISPR-based gene editing in human and mouse cardiomyocytes to repair Rbm20 mutations and analyze the outcome on TTN splicing. I showed that the splicing of TTN and other RBM20 target genes was successfully restored and consequently the DCM-phenotype was alleviated in juvenile mice. Secondly, I sought to analyze changes in the repertoire of TTN isoforms upon disease-relevant mutations in the RBM20 gene. To this end, I harnessed the power of long-read sequencing and split-pool barcoding to establish a single-cell sequencing method whereby I could measure changes in alternative splicing in single cells. Using this method, I analyzed Rbm20-dependent splicing in human cardiomyocytes which can be used to construct a comprehensive splice map of TTN and other isoforms that are expressed in patients with Rbm20 mutations. I envision that knowledge of such a splice map can be exploited for developing therapeutic strategies to revert aberrant alternative splicing in patients with DCM.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз

Цел на проекта

The sarcomeric protein titin (TTN) is mutated in 30% of patients with familial dilated cardiomyopathy (DCM), a common heart disease that is a global threat to the aging society. Aberrant alternative splicing of TTN is causally linked to DCM, therefore, to understand mechanisms of TTN splicing is crucial for developing therapeutic options, which so far do not exist for DCM. RBM20, a protein that regulates alternative splicing of TTN, is also mutated in many patients with an aggressive form of DCM. How RBM20 mediates alternative splicing of TTN is largely unknown. Due to its gigantic size, TTN mRNA is often precluded from transcriptome-wide single-cell analysis. Here, I aim to understand alternative splicing of TTN mediated by RBM20 on a single-cell level. To this end, I propose to develop a method for the detection of all transcript isoforms that can be produced by the 364 exons of TTN. Using this tool, termed TITIN-seq, together with complementary stem cell-based assays, I seek to analyze changes in the repertoire of TTN isoforms upon disease-relevant mutations in the RBM20 gene. Moreover, TITIN-seq is used to identify novel splice regulators of TTN, which, together with RBM20, could complete the picture of alternative splicing of TTN. The overarching goal is to construct a comprehensive map of TTN splicing by integrating data of all its isoforms in single cells and its regulatory proteins. I envision that knowledge of such a splice map can be exploited for developing therapeutic strategies to revert aberrant splicing of TTN in patients with DCM.

Оригинален текст от CORDIS (на английски).

Участници

EUROPEAN MOLECULAR BIOLOGY LABORATORY · HeidelbergКоординаторГермания
BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY · STANFORDСъединени щати