Peterson M L, Perry R P
Institue for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Mol Cell Biol. 1989 Feb;9(2):726-38. doi: 10.1128/mcb.9.2.726-738.1989.
The relative abundance of the mRNAs encoding the membrane (mu m) and secreted (mu s) forms of immunoglobulin mu heavy chain is regulated during B-cell maturation by a change in the mode of RNA processing. Current models to explain this regulation involve either competition between cleavage-polyadenylation at the proximal (mu s) poly(A) site and cleavage-polyadenylation at the distal (mu m) poly(A) site [poly(A) site model] or competition between cleavage-polyadenylation at the mu s poly(A) site and splicing of the C mu 4 and M1 exons, which eliminates the mu s site (mu s site-splice model). To test certain predictions of these models and to determine whether there is a unique structural feature of the mu s poly(A) site that is essential for regulation, we constructed modified mu genes in which the mu s or mu m poly(A) site was replaced by other poly(A) sites and then studied the transient expression of these genes in cells representative of both early- and late-stage lymphocytes. Substitutions at the mu s site dramatically altered the relative usage of this site and caused corresponding reciprocal changes in the usage of the mu m site. Despite these changes, use of the proximal site was still usually higher in plasmacytomas than in pre-B cells, indicating that regulation does not depend on a unique feature of the mu s poly(A) site. Replacement of the distal (mu m) site had no detectable effect on the usage of the mu s site in either plasmacytomas or pre-B cells. These findings are inconsistent with the poly(A) site model. In addition, we noted that in a wide variety of organisms, the sequence at the 5' splice junction of the C mu 4-to-M1 intron is significantly different from the consensus 5' splice junction sequence and is therefore suboptimal with respect to its complementary base pairing with U1 small nuclear RNA. When we mutated this suboptimal sequence into the consensus sequence, the mu mRNA production in plasmacytoma cells was shifted from predominantly mu s to exclusively mu m. This result unequivocally demonstrated that splicing of the C mu 4-to-M1 exon is in competition with usage of the mu s poly(A) site. A key feature of this regulatory phenomenon appears to be the appropriately balanced efficiencies of these two processing reactions. Consistent with predictions of the mu s site-splice model, B cells were found to contain mu m precursor RNA that had undergone the C mu 4-to-M1 splice but had not yet been polyadenylated at the mu m site.
编码免疫球蛋白μ重链膜型（μm）和分泌型（μs）的mRNA的相对丰度，在B细胞成熟过程中通过RNA加工模式的改变受到调控。目前用于解释这种调控的模型，要么涉及近端（μs）聚腺苷酸化位点的切割 - 聚腺苷酸化与远端（μm）聚腺苷酸化位点的切割 - 聚腺苷酸化之间的竞争[聚腺苷酸化位点模型]，要么涉及μs聚腺苷酸化位点的切割 - 聚腺苷酸化与Cμ4和M1外显子的剪接之间的竞争，后者会消除μs位点（μs位点 - 剪接模型）。为了检验这些模型的某些预测，并确定μs聚腺苷酸化位点是否存在对调控至关重要的独特结构特征，我们构建了修饰的μ基因，其中μs或μm聚腺苷酸化位点被其他聚腺苷酸化位点取代，然后研究这些基因在代表早期和晚期淋巴细胞的细胞中的瞬时表达。在μs位点进行替换显著改变了该位点的相对使用情况，并导致μm位点的使用发生相应的反向变化。尽管有这些变化，但浆细胞瘤中近端位点的使用通常仍高于前B细胞，这表明调控并不依赖于μs聚腺苷酸化位点的独特特征。替换远端（μm）位点对浆细胞瘤或前B细胞中μs位点的使用没有可检测到的影响。这些发现与聚腺苷酸化位点模型不一致。此外，我们注意到在多种生物体中，Cμ4至M1内含子5'剪接连接处的序列与共有5'剪接连接序列有显著差异，因此就其与U1小核RNA的互补碱基配对而言是次优的。当我们将这个次优序列突变为共有序列时，浆细胞瘤细胞中的μmRNA产生从主要是μs转变为仅为μm。这一结果明确表明，Cμ4至M1外显子的剪接与μs聚腺苷酸化位点的使用存在竞争。这种调控现象的一个关键特征似乎是这两种加工反应的效率得到了适当平衡。与μs位点 - 剪接模型的预测一致，发现B细胞含有经历了Cμ4至M1剪接但尚未在μm位点进行聚腺苷酸化的μm前体RNA。
