Intron function in the nonsense-mediated decay of beta-globin mRNA: indications that pre-mRNA splicing in the nucleus can influence mRNA translation in the cytoplasm.

Generally, mRNAs that prematurely terminate translation are abnormally low in abundance. In the case of mammalian cells, nonsense codons most often mediate a reduction in the abundance of newly synthesized, nucleus-associated mRNA by a mechanism that is not well understood. With the aim of defining cis-acting sequences that are important to the reduction process, the effects of particular beta-globin gene rearrangements on the metabolism of beta-globin mRNAs harboring one of a series of nonsense codons have been assessed. Results indicate that nonsense codons located 54 bp or more upstream of the 3'-most intron, intron 2, reduce the abundance of nucleus-associated mRNA to 10-15% of normal without altering the level of either of the two introns within pre-mRNA. The level of cytoplasmic mRNA is also reduced to 10-15% of normal, indicating that decay does not take place once the mRNA is released from an association with nuclei into the cytoplasm. A nonsense codon within exon 2 that does not reduce mRNA abundance can be converted to the type that does by (1) inserting a sufficiently large in-frame sequence immediately upstream of intron 2 or (2) deleting and reinserting intron 2 a sufficient distance downstream of its usual position. These findings indicate that only those nonsense codons located more than 54 bp upstream of the 3'-most intron reduce beta-globin mRNA abundance, which is remarkably consistent with which nonsense codons within the triosephosphate isomerase (TPI) gene reduce TPI mRNA abundance. We propose that the 3'-most exon-exon junction of beta-globin mRNA and, possibly, most mRNAs is marked by the removal of the 3'-most intron during pre-mRNA splicing and that the "mark" accompanies mRNA during transport to the cytoplasm. When cytoplasmic ribosomes terminate translation more than 54 nt upstream of the mark during or immediately after transport, the mRNA is subjected to nonsense-mediated decay. The finding that deletion of beta-globin intron 2 does not appreciably alter the effect of any nonsense codon on beta-globin mRNA abundance suggests that another cis-acting sequence functions in nonsense-mediated decay comparably to intron 2, at least in the absence of intron 2, possibly as a fail-safe mechanism. The analysis of deletions and insertions indicates that this sequence resides within the coding region and can be functionally substituted by intron 2.     

Cytoplasmic mRNA for human triosephosphate isomerase is immune to nonsense-mediated decay despite forming polysomes

Nonsense codons between position 14 within the first exon and position 193 within the penultimate exon of the human gene for triosephosphate isomerase reduce mRNA abundance to 25% of normal. The reduction in abundance is due to the decay of newly synthesized mRNA that copurifies with nuclei. TPI mRNA that copurifies with cytoplasm is immune to decay. We show here that immunity is not due to the failure of nonsense-containing mRNA to form polysomes. This finding indicates that cytoplasmic mRNA, in contrast to nucleus-associated mRNA, may have lost one or more factors that are required for nonsense-mediated decay or gained one or more factors that confer immunity to nonsense-mediated decay.     

At Least One Intron Is Required for the Nonsense-Mediated Decay of Triosephosphate Isomerase mRNA: a Possible Link between Nuclear Splicing and Cytoplasmic Translation

Mammalian cells have established mechanisms to reduce the abundance of mRNAs that harbor a nonsense codon and prematurely terminate translation. In the case of the human triosephosphate isomerase (TPI gene), nonsense codons located less than 50 to 55 bp upstream of intron 6, the 3′-most intron, fail to mediate mRNA decay. With the aim of understanding the feature(s) of TPI intron 6 that confer function in positioning the boundary between nonsense codons that do and do not mediate decay, the effects of deleting or duplicating introns have been assessed. The results demonstrate that TPI intron 6 functions to position the boundary because it is the 3′-most intron. Since decay takes place after pre-mRNA splicing, it is conceivable that removal of the 3′-most intron from pre-mRNA “marks” the 3′-most exon-exon junction of product mRNA so that only nonsense codons located more than 50 to 55 nucleotides upstream of the “mark” mediate mRNA decay. Decay may be elicited by the failure of translating ribosomes to translate sufficiently close to the mark or, more likely, the scanning or looping out of some component(s) of the translation termination complex to the mark. In support of scanning, a nonsense codon does not elicit decay if some of the introns that normally reside downstream of the nonsense codon are deleted so the nonsense codon is located (i) too far away from a downstream intron, suggesting that all exon-exon junctions may be marked, and (ii) too far away from a downstream failsafe sequence that appears to function on behalf of intron 6, i.e., when intron 6 fails to leave a mark. Notably, the proposed scanning complex may have a greater unwinding capability than the complex that scans for a translation initiation codon since a hairpin structure strong enough to block translation initiation when inserted into the 5′ untranslated region does not block nonsense-mediated decay when inserted into exon 6 between a nonsense codon residing in exon 6 and intron 6.     

Nonsense-mediated decay of glutathione peroxidase 1 mRNA in the cytoplasm depends on intron position

mRNA for glutathione peroxidase 1 (GPx1) is subject to cytoplasmic nonsense-mediated decay (NMD) when the UGA selenocysteine (Sec) codon is recognized as nonsense. Here, we demonstrate by moving the sole intron of the GPx1 gene that either the Sec codon or a TAA codon in its place elicits NMD when located >/=59 bp but not 相似文献   

Translation to near the distal end of the penultimate exon is required for normal levels of spliced triosephosphate isomerase mRNA.

The translation of human triosephosphate isomerase (TPI) mRNA normally terminates at codon 249 within exon 7, the final exon. Frameshift and nonsense mutations within the TPI gene that cause translation to terminate prematurely at or upstream of codon 189, within exon 6, result in a decreased level of TPI mRNA (I.O. Daar and L.E. Maquat, Mol. Cell. Biol. 8:802-813, 1988). For all mutations in this group, the decrease is to the same extent, i.e., to approximately 20% of the normal level. We show here that a second group of nonsense mutations that cause translation to terminate prematurely at or downstream of codon 208, in exon 6, did not affect TPI mRNA abundance. Deletion analysis demonstrated that the abundance of translationally active TPI mRNA is a function of both the distance and the polarity of the nonsense codon relative to the final intron in TPI pre-mRNA. Our results indicate that if translating ribosomes are unable to progress to at least a certain position within the penultimate exon relative to the final intron, then the level of the corresponding mRNA will be abnormally low. Studies inhibiting RNA synthesis with dactinomycin demonstrated that a block in translation does not affect the half-life of mature TPI mRNA. The simplest interpretation of our data is that the translation of TPI mRNA in the cytoplasm facilitates the splicing of TPI pre-mRNA or the transport of TPI mRNA across the nuclear envelope or both.     

Introns are cis effectors of the nonsense-codon-mediated reduction in nuclear mRNA abundance.

The translation of human triosephosphate isomerase (TPI) mRNA normally terminates at codon 249 within exon 7, the final exon. Frameshift and nonsense mutations of the type that cause translation to terminate prematurely at or upstream of codon 189 within exon 6 reduce the level of nuclear TPI mRNA to 20 to 30% of normal by a mechanism that is not a function of the distance of the nonsense codon from either the translation initiation or termination codon. In contrast, frameshift and nonsense mutations of another type that cause translation to terminate prematurely at or downstream of codon 208, also within exon 6, have no effect on the level of nuclear TPI mRNA. In this work, quantitations of RNA that derived from TPI alleles in which nonsense codons had been generated between codons 189 and 208 revealed that the boundary between the two types of nonsense codons resides between codons 192 and 195. The analysis of TPI gene insertions and deletions indicated that the positional feature differentiating the two types of nonsense codons is the distance of the nonsense codon upstream of intron 6. For example, the movement of intron 6 to a position downstream of its normal location resulted in a concomitant downstream movement of the boundary between the two types of nonsense codons. The analysis of intron 6 mutations indicated that the intron 6 effect is stipulated by the 88 nucleotides residing between the 5' and 3' splice sites. Since the deletion of intron 6 resulted in only partial abrogation of the nonsense codon-mediated reduction in the level of TPI mRNA, other sequences within TPI pre-mRNA must function in the effect. One of these sequences may be intron 2, since the deletion of intron 2 also resulted in partial abrogation of the effect. In experiments that switched introns 2 and 6, the replacement of intron 6 with intron 2 was of no consequence to the effect of a nonsense codon within either exon 1 or exon 6. In contrast, the replacement of intron 2 with intron 6 was inconsequential to the effect of a nonsense codon in exon 6 but resulted in partial abrogation of a nonsense codon in exon 1.