We previously reported a 40-transcripts signature marking the normal mucosa to colorectal adenocarcinoma transition. Eight of these mRNAs also showed splicing alterations, including a specific intron 3 retention in tissue metalloprotease inhibitor I (TIMP1), which decreased during the early steps of colorectal cancer progression. To decipher the mechanism of intron 3 retention/splicing, we first searched for putative RNA binding protein binding sites onto the TIMP1 sequence. We identified potential serine arginine rich splicing factor 1 (SRSF1) and heterogeneous nuclear RiboNucleoProtein A1 (hnRNPA1) binding sites at the end of intron 3 and the beginning of exon 4, respectively. RNA immunoprecipitation showed that hnRNPA1, but not SRSF1 could bind to the corresponding region in TIMP1 pre-mRNA in live cells. Furthermore, using a TIMP1-based ex vivo minigene approach, together with a plasmon resonance in vitro RNA binding assay, we confirmed that hnRNPA1 could indeed bind to wild type TIMP1 exon 4 pre-mRNA and control TMP1 intron 3 splicing, the interaction being abolished in presence of a mutant sequence that disrupted this site. These results indicated that hnRNPA1, upon binding to TIMP1 exon 4, was a positive regulator of intron 3 splicing. We propose that this TIMP1-intron 3 + transcript belongs to the class of nuclear transcripts with "detained" introns, an abundant molecular class, including in cancer.

中文翻译：

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TIMP1内含子3保留是hnRNPA1控制的结肠癌进展的标志。

我们以前报道过40个转录物的标记，标志着正常的粘膜向大肠腺癌过渡。这些mRNA中的八个还显示了剪接改变，包括组织金属蛋白酶抑制剂I（TIMP1）中特定的内含子3保留，该保留在结直肠癌进展的早期阶段减少。若要破译内含子3保留/剪接的机制，我们首先在TIMP1序列上搜索推定的RNA结合蛋白结合位点。我们确定了潜在的富含丝氨酸精氨酸的剪接因子1（SRSF1）和异质核RiboNucleoProtein A1（hnRNPA1）结合位点分别在内含子3的末端和外显子4的开头。RNA免疫沉淀显示，hnR​​NPA1而非SRSF1可以与活细胞TIMP1 pre-mRNA中的相应区域结合。此外，使用基于TIMP1的离体小基因方法以及等离子体共振体外RNA结合测定法，我们证实hnRNPA1确实可以与野生型TIMP1外显子4 pre-mRNA结合并控制TMP1内含子3剪接，在存在时相互作用被取消破坏该位点的突变序列。这些结果表明，hnRNPA1与TIMP1外显子4结合后，是内含子3剪接的正调控子。我们建议该TIMP1内含子3 +转录本属于具有“被拘留的”内含子的核转录本类别，内含子是一个丰富的分子类别，包括在癌症中。在破坏该位点的突变序列的存在下，相互作用被取消。这些结果表明，hnRNPA1，与TIMP1外显子4结合后，是内含子3剪接的阳性调节子。我们建议该TIMP1内含子3 +转录本属于具有“被拘留”内含子的核转录本类别，内含子是一个丰富的分子类别，包括在癌症中。在破坏该位点的突变序列的存在下，相互作用被取消。这些结果表明，hnRNPA1，与TIMP1外显子4结合后，是内含子3剪接的阳性调节子。我们建议该TIMP1内含子3 +转录本属于具有“被拘留”内含子的核转录本类别，内含子是一个丰富的分子类别，包括在癌症中。