커뮤니케이션 생물학 5권, 기사 번호: 987(2022) 이 기사 인용
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대체 접합은 골격근 발달 및 병리와 관련된 RNA 처리 메커니즘입니다. 근육 질환은 기계적 힘과 RNA 처리 사이의 상호 연결을 조사하도록 유도하는 기계생물학의 접합 변경 및 변화를 나타냅니다. 우리는 근육 세포를 스트레칭한 후 딥 RNA 시퀀싱을 수행했습니다. 첫째, 우리는 근육 기능과 전사에 관여하는 단백질을 코딩하는 유전자의 전사 변화를 밝혀냈습니다. 둘째, 우리는 수많은 기계적 민감성 유전자가 스트레칭에 반응하여 활성화되는 MAPK 경로의 일부임을 관찰했습니다. 셋째, 골격근 세포를 스트레칭하면 교대로 접합된 카세트 엑손의 비율과 그 포함이 증가한다는 사실이 밝혀졌습니다. 넷째, 우리는 세린과 아르기닌이 풍부한 단백질이 다른 RNA 결합 단백질보다 더 강한 전사 변화를 나타내며 SRSF4 인산화가 기계 민감성임을 입증했습니다. SRSF4를 기계적 변환, 전사 및 접합 사이의 누화에 기여할 수 있는 기계 민감성 RNA 결합 단백질로 식별하면 근육 질환, 특히 병인이 알려지지 않은 질환에 대한 새로운 통찰력을 잠재적으로 밝힐 수 있습니다.
Alternative splicing은 단백질 다양성을 확장하는 RNA 처리 메커니즘으로 고등 진핵생물에서 매우 널리 퍼져 있으며 인간 유전자의 95% 이상이 Alternative splicing을 겪고 있습니다1. 골격근에서는 출생 후 광범위한 대체 접합 전환이 발생하여 수축 장치3,4,5,6의 성숙에 기여합니다. 골격근은 발달을 위해 분자 메커니즘과 기계적 특성에 의존하는 복잡한 조직입니다. 근육 수축 장치는 근절, 횡세관 및 코스타메어로 구성되며, 이들은 기계적 변환이라고 알려진 과정에서 근육 세포와 핵에서 원형질막으로 힘을 함께 변환합니다. 흥미롭게도 다양한 근육 질환에서 성인의 대체 접합 패턴은 근육 손실, 위축 및 근육의 기능적 실패에 기여하는 태아 단계로 되돌아갑니다3,4,8,9. 따라서 골격근의 분자 전이는 조직의 적절한 발달에 중요합니다.
골격근은 발달 중에 분자 전이를 겪는 것 외에도 기계적 힘에 반응하고 수축을 통해 조화로운 힘의 폭발을 생성해야 합니다. 근육 세포는 특히 기계변환에 민감합니다10. 주변 환경의 강성은 세포 신장과 분화를 제어하며, 이는 효율적인 근육 수축에 중요합니다. 건강한 사람에 비해 근이영양증을 앓고 있는 사람의 근육 세포는 더 뻣뻣하며 이는 적절한 수축을 방해하여 궁극적으로 근육 소모로 이어지는 것으로 생각됩니다11,12,13.
전체적인 잘못된 접합 또는 개별 대체 접합 이벤트의 잘못된 조절로 인해 적절한 근육 기능이 손실된다는 것이 광범위하게 입증되었습니다2,3,4,6,14,15. Duchenne 근이영양증(스플라이싱 조절 장애)이 있는 마우스는 근육의 물리적 변화가 분자 반응과 연관되어 있음을 나타내는 기계적 민감성 신호 전달 경로의 변경을 나타냅니다16. 이 아이디어를 확장하면 근이영양증이 있는 노화된 쥐는 스플라이스 전환 요법과 운동을 모두 포함하는 개입에 대한 반응으로 피로가 감소한 것으로 나타났습니다17. 이 연구는 근육 질환, 접합 잘못된 조절 및 근육의 기계적 변화 사이의 연관성을 확립합니다. 그러나 기계적 힘과 대체 접합이 근육 항상성을 유지하기 위해 어떻게 협력하는지, 그리고 근육 질환의 발병에 어느 정도 기여하는지는 알려져 있지 않습니다.
90% of them mapping to the mm10 mouse genome (Supplementary Data 1). The high mapping rate indicates appropriate quality of our sequencing data. We first confirmed that several myogenesis markers were not expressed in the myoblast samples and were highly induced in the differentiated cells (Supplementary Fig. 1). We next performed principal component analysis (PCA) to cluster samples based on gene expression for both myoblasts (Fig. 1a) and differentiated cells (Fig. 1b). The non-stretched myoblasts segregated together with two distinct subclusters for the 1 h stretched samples and the 6 h stretched samples (Fig. 1a, dark green and dark blue respectively). The 3 h stretched myoblasts segregated closer to the non-stretched samples (Fig. 1a, dark purple). In the differentiated cells, all the non-stretched samples segregated together and we observed three distinct groups for the different stretching time points demonstrating that varying times of stretching resulted in distinct gene expression changes (Fig. 1b, dark green, dark purple, and dark blue). One of the 1 h stretched samples did not segregate closely with the other replicates but remained more similar to them than to samples at other time points. This sample passed other quality controls and was therefore included with all samples in the analysis described in the results. Post hoc tests excluding this sample were performed and did not change the conclusions of the study./p>1.50 (upregulated, up) or fold change < −1.50 (downregulated, down). e, f. Correlation plot between the gene expression changes (expressed as log2foldchange) as measured by qPCR assays and those observed in the RNA-seq studies in myoblasts and differentiated cells. The qPCR graphs for the individual genes included in the correlation plots are shown in Supplementary Fig. 2 (myoblasts) and Supplementary Fig. 3 (differentiated cells)./p>1.5 for upregulated genes and a fold change <−1.5 for downregulated genes and an adjusted p-value < 0.05. Two of the genes assayed in both myoblasts and differentiated cells were the connective tissue growth factor (Ctgf) and the cysteine-rich angiogenic inducer 61 (Cyr61) which are both well-established mechanosensitive genes26,27,28. Ctgf and Cyr61 mRNAs were upregulated after 1 h of stretching in myoblasts and after 1 h and 3 h of stretching in differentiated cells indicating successful cellular stretching (Supplementary Figs. 2, 3). After 6 h of stretching, Ctgf and Cyr61 mRNAs were not upregulated in either myoblasts or differentiated cells (Supplementary Figs. 2, 3) suggesting that the cells become accustomed to the long mechanical stimulus which has been previously reported28./p> 10. In myoblasts, we observed an increase in the total number of splicing events as the cells were stretched for longer periods of time (Fig. 4a, left). Whereas in differentiated cells, the total number of splicing events increased from 1 h to 3 h and slightly decreased from 3 h to 6 h (Fig. 4a, right)./p> 10 between stretched and non-stretched samples. The ΔPSI was defined as the difference between the PSI in stretched samples and the PSI in the non-stretched controls. PSI percent spliced in./p>44% of splicing events at all time points were cassette exons (Fig. 5b). Second, we found that as cells were stretched, the proportion of cassette exons being alternatively spliced increased over time while the proportion of retained introns decreased (except for the transition from 3 h to 6 h in differentiated cells) (Fig. 5b). We focused the rest of our analysis on cassette exons since these were the most prevalent type of splicing event./p> 10, blue) or more exclusion (ΔPSI < −10, red) of the alternatively spliced region upon stretching. The numbers between parentheses indicate the number of splicing events. The ΔPSI was defined as the difference between the PSI in stretched samples and the PSI in the non-stretched controls. PSI percent spliced in./p> 10 (stretching induces inclusion) increases over stretching time (Supplementary Fig. 8a, left). In differentiated cells, we observed a more drastic trend in the same direction (Supplementary Fig. 8a, right). Since we focused most of our analysis on cassette exons we also determined if stretching induced more skipping or inclusion specifically of cassette exons. In myoblasts, the proportion of cassette exons with ΔPSI <−10 (stretching induces exclusion) increases slightly over stretching time (Fig. 5e, top). In differentiated cells, we observed an opposite trend with longer stretching times inducing more inclusion of cassette exons (Fig. 5e, bottom)./p> 95, r28S:18 S > 2.8 and RNA integrated number (RIN) > 8.4. Kapa mRNA stranded method was used for library preparation. All samples were pooled, and the library was first run on an Illumina MiSeq Nano to verify sequencing quality. After quality verification, the library was run on one flow cell over four lanes on a NovaSeq 6000S4 sequencer in a paired end 2 × 100 cycle run at the High Throughput Sequencing Core at The University of North Carolina at Chapel Hill./p>1.50 for upregulated genes, or fold change <−1.50 for downregulated genes). Significant changes in gene expression for myoblasts are in Supplementary Data 2 and for differentiated cells are in Supplementary Data 3. To determine differential alternative splicing in response to stretching, reads were aligned to the Gencode mm10 genome and Gencode vM20 transcriptome that contained reference chromosomes, scaffolds, assembly patches, and haplotypes. Differential splicing was determined using MISO and datasets were created that contained the MISO summary counts of all samples at each timepoint35. Those datasets were then used to compare the stretched samples to the non-stretched controls at each timepoint. Events were filtered by total read depth ≥15 and either inclusion reads ≥5 or exclusion reads ≥5. After filtering, events were considered alternatively spliced if |ΔPSI| (|PSIstretch–PSIno stretch|) >10 and the Wilcox p-value (stretch versus no stretch) ≤0.05. Significant changes in alternative splicing for myoblasts are in Supplementary Data 4 and those for differentiated cells are in Supplementary Data 5. In the excel files the ΔPSI values are shown in a scale from 0 to 1 (for inclusion) or 0 to −1 (for exclusion)./p>3.0.CO;2-C" data-track-action="article reference" href="https://doi.org/10.1002%2F%28SICI%291097-0177%28199808%29212%3A4%3C495%3A%3AAID-AJA3%3E3.0.CO%3B2-C" aria-label="Article reference 47" data-doi="10.1002/(SICI)1097-0177(199808)212:43.0.CO;2-C"Article CAS PubMed Google Scholar /p>
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