Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of thetd gene by recombinational marker rescue using plasmid or M13 subclones of thetd gene. Phage crosses using deletion mutants with known breakpoints in the 3′ end of thetd intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5′ and 3′ ends of the intron sequence, in accordance with the hypothesis that the 5′ and 3′ domains of the T4td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mappedtd mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within thetd gene.     

Identification of an intronic splicing regulatory element involved in auto‐regulation of alternative splicing of SCL33 pre‐mRNA

In Arabidopsis, pre‐mRNAs of serine/arginine‐rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis‐elements and trans‐acting proteins involved in regulating AS. Using a splicing reporter (GFP–intron–GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis‐elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis‐elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP–intron–GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto‐regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis‐element involved in AS of a plant SR gene, and elucidated a mechanism for auto‐regulation of AS of this intron.     

nMAT4, a maturase factor required for nad1 pre‐mRNA processing and maturation,is essential for holocomplex I biogenesis in Arabidopsis mitochondria

Group II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lower eukaryotes, but are particularly prevalent within mitochondria in plants, where they are present in many critical genes. The excision of plant mitochondrial introns is essential for respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self‐splicing ribozyme and its own intron‐encoded maturase protein. A hallmark of maturases is that they are intron‐specific, acting as cofactors that bind their intron‐containing pre‐RNAs to facilitate splicing. However, the degeneracy of the mitochondrial introns in plants and the absence of cognate intron‐encoded maturase open reading frames suggest that their splicing in vivo is assisted by ‘trans’‐acting protein factors. Interestingly, angiosperms harbor several nuclear‐encoded maturase‐related (nMat) genes that contain N‐terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. Here we show that nMAT4 (At1g74350) is required for RNA processing and maturation of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria. Seed germination, seedling establishment and development are strongly affected in homozygous nmat4 mutants, which also show modified respiration phenotypes that are tightly associated with complex I defects.     

YB‐1 orchestrates onset and resolution of renal inflammation via IL10 gene regulation

The Y‐box‐binding protein (YB)‐1 plays a non‐redundant role in both systemic and local inflammatory response. We analysed YB‐1‐mediated expression of the immune regulatory cytokine IL‐10 in both LPS and sterile inflammation induced by unilateral renal ischaemia–reperfusion (I/R) and found an important role of YB‐1 not only in the onset but also in the resolution of inflammation in kidneys. Within a decisive cis‐regulatory region of the IL10 gene locus, the fourth intron, we identified and characterized an operative YB‐1 binding site via gel shift experiments and reporter assays in immune and different renal cells. In vivo, YB‐1 phosphorylated at serine 102 localized to the fourth intron, which was paralleled by enhanced IL‐10 mRNA expression in mice following LPS challenge and in I/R. Mice with half‐maximal expression of YB‐1 (Yb1^+/?) had diminished IL‐10 expression upon LPS challenge. In I/R, Yb1^+/? mice exhibited ameliorated kidney injury/inflammation in the early‐phase (days 1 and 5), however showed aggravated long‐term damage (day 21) with increased expression of IL‐10 and other known mediators of renal injury and inflammation. In conclusion, these data support the notion that there are context‐specific decisions concerning YB‐1 function and that a fine‐tuning of YB‐1, for example, via a post‐translational modification regulates its activity and/or localization that is crucial for systemic processes such as inflammation.     

A non-directed, hydroxylamine-generated suppressor mutation in the P3 pairing region of the bacteriophage T4 td intron partially restores self-splicing capability

Hydroxylamine (HA) mutagenesis of an HA-induced splicing-defective bacteriophage T4 td intron mutant with a mutation in the intron P3 RNA pairing region was used to generate pseudorevertants. Because HA can only cause GC to AT transitions, the original mutant (H104A) could not undergo true reversion, yet the compensatory mutation on the opposite side of the P3 helix, which was complementary to the original H104A mutation, could occur. A pseudorevertant was isolated that contained both the original H104A mutation and the compensatory mutation HS9. By phenotypic and molecular genetic criteria, this double mutant (H104A-HS9) was shown to be able to undergo significant RNA splicing, thus confirming the existence and functional importance of the long-range P3 pairing region in this phage intron. The second-site suppressor mutation (HS9) was isolated by phage cross and also exhibited some self-splicing ability. A correlation exists between the strength of P3 helix Watson-Crick base pairing and the apparent level of splicing when wild-type, H104A, HS9, and H104A-HS9 are compared. This suggests that the primary role of the P3 RNA pairing region in the T4 td intron is structural in contributing to the critical RNA secondary structure.     

Molecular cloning of the T4 genome; organization and expression of the frd--DNA ligase region

Summary derivatives including the thymidylate synthetase (td) gene of T4 were selected by their ability to substitute for the thyA gene of E. coli. Two HindIII fragments of T4 DNA, but only one EcoRI fragment, are required for a functional td gene; one of the HindIII fragments includes a functional frd gene. The organisation of the EcoRI and HindIII fragments in the td region and their orientation with respect to the T4 genome have been deduced from genetic, physical, and functional evidence. The T4 genes can be transcribed from phage promoters and the T4td derivatives include genes specifying five T4 polypeptides. Three of these are identified as the products of the frd, td, and nrdA genes; two, neither of which appears to be the nrdB gene product, remain to be identified. Some td phages yield lysogens of thyA bacteria which are thymine-independent and some frd phages yield trimethoprim-resistant lysogens, indicating that the td and frd genes can be transcribed from included T4 DNA sequences. EcoRI fragments of DNA from the td and lig regions, used as probes, identified a single large HindIII fragment that joins the HindIII fragment carrying the DNA ligase gene to that carrying the td gene. Since this fragment, which must include genes coding for RNA ligase and polynucleotide kinase, could not be recovered in either phage or plasmid vectors, a derivative of it was used to identify the EcoRI fragments located between the td and DNA ligase genes. The order of these fragments within the T4 genome was deduced and all but one of them cloned in a vector. As none of these recombinants rescued T4 phage having mutations within the RNA ligase gene, the missing fragment may include this gene. Three adjacent EcoRI fragments, each of which has been cloned, are missing in a mutant of T4 deleted for the polynucleotide kinase gene.