Alternative Splicing Generates a Novel Truncated Cav1.2 Channel in Neonatal Rat Heart

L-type Ca_v1.2 Ca²⁺ channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Ca_v1.2 Ca²⁺ channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Ca_v1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Ca_v1.2_33L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Ca_v1.2 channels, Ca_v1.2_33L channels reduced the current density and altered the electrophysiological properties of the wild type Ca_v1.2 channels. Interestingly, the truncated 3.5-domain Ca_v1.2_33L channels also yielded a dominant negative effect on Ca_v1.3 channels, but not on Ca_v3.2 channels, suggesting that Ca_vβ subunits is required for Ca_v1.2_33L regulation. A biochemical study provided evidence that Ca_v1.2_33L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Ca_v1.2_33L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Ca_v1.2 channels. Moreover, the human Ca_v1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Ca_v1.2 channel. An electrophysiological study showed that human Ca_v1.2_33L channel is a functional channel but conducts Ca²⁺ ions at a much lower level.     

鉴定9个新的RHD基因mRNA可变剪接体

为了研究各种RHD基因mRNA可变剪接体的基因结构, 应用逆转录聚合酶链反应(RT-PCR)检测正常人脐血样本RHD mRNA, 对RHD cDNA进行TA克隆和序列分析, 对各可变剪接体的剪接位点进行DNA序列分析, 并将RHD mRNA进行表达序列标签(ESTs)分析。结果在28个阳性克隆中, 除全长RHD cDNA外, 共检测到12种(包括9种新的)RHD可变剪接体, 发现外显子遗漏、5′和3′剪接位点变异3种剪接形式, 涉及外显子2~9, 其中6种新的剪接体同时存在RHD和RHCE基因同源杂交现象。ESTs分析还检索到内含子保留形式的剪接体。研究表明, RHD基因mRNA存在复杂的可变剪接机制, 除已报道的剪接体外, 检测到9种新的RHD可变剪接体, 并发现了可变剪接和同源杂交并存现象。     

中国人苯丙氨酸羟化酶基因的一个新的切接位点突变的鉴定

本文鉴定了苯丙氨酸羟化酶基因355位密码子上的一个新的错义突变Q355 H, 此突变异致谷氨酰胺变成了组氨酸。此突变位点恰位于外显子10和内含子11的交界处, 因此将引起mRNA形成过程中的剪接错误而产生异常的mR NA。Q355H的鉴定为一例苯丙酮尿症胎儿的产前诊断提供了理论依据。 Abstract:A novel missense mutation at code 355 of phenylalanine hydroxlase gene was identified,this mutation caused the substitution of Gln 355 for His 355.The mutant site was at the boundary of exon 10 and intro 11 and might cause splicing errors during RNA processing,Which could result in abnormal mRNA.Identification of Q355H provided a theortic evidence for prenatal diagnosis of a fetus with PKU.     

人受精促进肽受体基因（TCP11b）的剪接、克隆和差别表达

运用同源比较和PCR法 ,从人睾丸组织中分离了人受精促进肽受体TCP11基因的一个新的剪切体TCP11b ,它编码 5 0 3个氨基酸的蛋白质 ,与TCP11a相比 ,在基因组的 5′端存在复杂的外显子剪接现象。运用荧光原位杂交 (FISH)方法 ,显示该基因定位到人染色体 6p2 1。Northern杂交及多组织RT PCR的结果显示该转录本在正常睾丸中表达 ,而其他组织、无精症患者及胎儿睾丸组织中未见该基因的表达。该结果结合mTcp 11功能的提示 ,TCP11b这种转录本对精子发生和人受精过程可能起重要作用。     

A Novel,Universally Active C-terminal Protein Degradation Signal Generated by Alternative Splicing

Proteome integrity is crucial for cellular homeostasis and adaptation to stress conditions such as hypoxia. One mechanism for rapid adaptation of the proteome in response to changing environmental signals is alternative splicing. In addition to generating different protein isoforms, alternative splicing is also capable of controlling total protein levels by the regulated synthesis of non-productive mRNA isoforms. The hypoxia-induced isoform E of the tumor suppressor MAX is produced by retention and translation of the last intron. This leads to an alternative C-terminus that harbors a potent C-degron, the isoE degron. Strikingly, the isoE degron represents a universal protein degradation signal that is not only functional in mammalian cells, but also in yeast and even in bacteria. Essential for efficient protein decay is a conserved (F/W)xxW motif. Degradation of isoE tagged proteins is mediated by the proteasome in eukaryotes and Lon protease in bacteria. Thus, the isoE degron is a broadly applicable and highly efficient tool in protein analyses.     

Targeting the Spleen as an Alternative Site for Hematopoiesis

Bone marrow is the main site for hematopoiesis in adults. It acts as a niche for hematopoietic stem cells (HSCs) and contains non‐hematopoietic cells that contribute to stem cell dormancy, quiescence, self‐renewal, and differentiation. HSC also exist in resting spleen of several species, although their contribution to hematopoiesis under steady‐state conditions is unknown. The spleen can however undergo extramedullary hematopoiesis (EMH) triggered by physiological stress or disease. With the loss of bone marrow niches in aging and disease, the spleen as an alternative tissue site for hematopoiesis is an important consideration for future therapy, particularly during HSC transplantation. In terms of harnessing the spleen as a site for hematopoiesis, here the remarkable regenerative capacity of the spleen is considered with a view to forming additional or ectopic spleen tissue through cell engraftment. Studies in mice indicate the potential for such grafts to support the influx of hematopoietic cells leading to the development of normal spleen architecture. An important goal will be the formation of functional ectopic spleen tissue as an aid to hematopoietic recovery following clinical treatments that impact bone marrow. For example, expansion or replacement of niches could be considered where myeloablation ahead of HSC transplantation compromises treatment outcomes.