To understand the role of U2 RNA structure in pre-mRNA splicing we have characterized several cold-sensitive mutations in an essential stem-loop of yeast U2. Although mutant U2 is stable in vivo after a shift to restrictive temperature, splicing is rapidly inhibited, suggesting a direct effect on U2 function rather than U2 synthesis or snRNP assembly. Splicing complexes form at 23~ in both mutant and wild-type extracts; however, stable association of mutant U2 snRNPs with pre-mRNA in vitro is inefficient at 15~ a temperature permissive for spliceosome assembly in wild-type extracts, indicating that the cold-sensitive defect is in U2 snRNP association with the assembling spliceosome. In vivo RNA structure probing reveals that the bulk of U2 RNA is misfolded in the mutants, even at permissive temperature. We propose that U2 stem-loop IIa is recognized by an assembly factor that assists U2 snRNP binding to pre-mRNA and that the cold sensitivity is due to a critical deficiency of correctly folded U2 for spliceosome assembly at low temperatures. Evolutionary conservation of the potential to form an interfering alternative RNA structure suggests the possibility that splicing could be regulated negatively at an early step by control of U2 snRNA conformation.

Mutations in stem-loop Ila of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.RNA-RNA interactions between small nuclear RNAs (snRNAs) or between snRNAs and the pre-mRNA play critical roles in the accuracy and efficiency of splicing (for reviews, see references 15 and 16). Not all of these interactions are established simultaneously, nor do they persist once established. Rather, interactions are formed, modified, disrupted, and replaced during spliceosome assembly and splicing. The dynamic relationship between U4 and U6 has been known for some time (15, 16), and more recently it has been shown that U2 also interacts with U6 (11,26,47 Structure-function studies in this region of U2 show that only one of the structures, that containing stem-loop Ila, is absolutely essential for growth, arguing that the potential to form the others must be conserved for an accessory or overspecified function (2). Direct probing of yeast U2 structure in vivo confirms that the bulk of U2 in the cell adopts the essential structure (2); however, the alternative structure can form under certain circumstances, suggesting that this region of U2 snRNA is dynamic (49).An unanticipated phenotype associated with single base changes that destabilize the essential structure is cold-sensitive growth (2). Cold sensitivity is also observed in cell splicing extracts, allowing the demonstration of a role for stem Ila in the critical step of assembly of U2 snRNPs into the spliceosome in vitro (49). Structure probing experiments show that the bulk of U2 snRNA is misfolded in the cold-sensitive mutants, so that the RNA adopts the other phylogenetically conserved structure. Surprisingly, the misfolded form predominates at both permissive and restrict...

The vulnerability of the human brain to injury following just a few minutes of oxygen deprivation with submergence contrasts markedly with diving mammals, such as Weddell seals (Leptonychotes weddellii), which can remain underwater for more than 90 min while exhibiting no neurological or behavioural impairment. This response occurs despite exposure to blood oxygen levels concomitant with human unconsciousness. To determine whether such aquatic lifestyles result in unique adaptations for avoiding ischaemic-hypoxic neural damage, we measured the presence of circulating (haemoglobin) and resident (neuroglobin and cytoglobin) oxygen-carrying globins in the cerebral cortex of 16 mammalian species considered terrestrial, swimming or diving specialists. Here we report a striking difference in globin levels depending on activity lifestyle. A nearly 9.5-fold range in haemoglobin concentration (0.17-1.62 g Hb 100 g brain wet wt K1) occurred between terrestrial and deep-diving mammals; a threefold range in resident globins was evident between terrestrial and swimming specialists. Together, these two globin groups provide complementary mechanisms for facilitating oxygen transfer into neural tissues and the potential for protection against reactive oxygen and nitrogen groups. This enables marine mammals to maintain sensory and locomotor neural functions during prolonged submergence, and suggests new avenues for averting oxygen-mediated neural injury in the mammalian brain.

Mammalian L1 (long interspersed repeated DNA, LINE-1) retrotransposons consist of a 5′ untranslated region (UTR) with regulatory properties, two protein encoding regions (ORF I, ORF II, which encodes a reverse transcriptase) and a 3′ UTR. L1 elements have been evolving in mammals for >100 million years and this process continues to generate novel L1 subfamilies in modern species. Here we characterized the youngest known subfamily in Rattus norvegicus, L1mlvi2, and unexpectedly found that this element has a dual ancestry. While its 3′ UTR shares the same lineage as its nearest chronologically antecedent subfamilies, L13 and L14, its ORF I sequence does not. The L1mlvi2 ORF I was derived from an ancestral ORF I sequence that was the evolutionary precursor of the L13 and L14 ORF I. We suggest that an ancestral ORF I sequence was recruited into the modern L1mlvi2 subfamily by recombination that possibly could have resulted from template strand switching by the reverse transcriptase during L1 replication. This mechanism could also account for some of the structural features of rodent L1 5′ UTR and ORF I sequences including one of the more dramatic features of L1 evolution in mammals, namely the repeated acquisition of novel 5′ UTRs.