Schott Johanna, Reitter Sonja, Lindner Doris, Grosser Jan, Bruer Marius, Shenoy Anjana, Geiger Tamar, Mathes Arthur, Dobreva Gergana, Stoecklin Georg
Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
Center for Molecular Biology of Heidelberg University (ZMBH), German Cancer Research Center (DKFZ)-ZMBH Alliance, Heidelberg, Germany.
Nat Methods. 2021 Sep;18(9):1068-1074. doi: 10.1038/s41592-021-01250-z. Epub 2021 Sep 3.
In general, mRNAs are assumed to be loaded with ribosomes instantly upon entry into the cytoplasm. To measure ribosome density (RD) on nascent mRNA, we developed nascent Ribo-Seq by combining Ribo-Seq with progressive 4-thiouridine labeling. In mouse macrophages, we determined experimentally the lag between the appearance of nascent mRNA and its association with ribosomes, which was calculated to be 20-22 min for bulk mRNA. In mouse embryonic stem cells, nRibo-Seq revealed an even stronger lag of 35-38 min in ribosome loading. After stimulation of macrophages with lipopolysaccharide, the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments, which gives rise to distorted RD measurements under conditions where mRNA amounts are far from steady-state expression. As a result, we demonstrate that transcriptional changes affect RD in a passive way.
一般来说，人们认为mRNA一进入细胞质就会立即被核糖体装载。为了测量新生mRNA上的核糖体密度（RD），我们通过将核糖体图谱测序（Ribo-Seq）与渐进性4-硫尿苷标记相结合，开发了新生核糖体图谱测序（nascent Ribo-Seq）。在小鼠巨噬细胞中，我们通过实验确定了新生mRNA出现与其与核糖体结合之间的延迟，经计算，大量mRNA的延迟时间为20 - 22分钟。在小鼠胚胎干细胞中，新生核糖体图谱测序显示核糖体装载的延迟甚至更长，为35 - 38分钟。在用脂多糖刺激巨噬细胞后，细胞质mRNA与已翻译mRNA之间的延迟导致输入片段与核糖体保护片段之间的解偶联，这在mRNA量远非稳态表达的条件下导致核糖体密度测量结果失真。因此，我们证明转录变化以被动方式影响核糖体密度。
