We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m 6 A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3 ′ -most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m 6 A and >40% of all m 6 A in mRNA are present in 3 ′ untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m 6 A sites around stop codons. Moreover, m 6 A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m 6 A density peaks early in the 3 ′ UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m 6 A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m 6 A modification in regulating proximal alternative polyA choice.

Zebra finches are widely used for studying the basic biology of vocal learning. The inability to introduce genetic modifications in these animals has substantially limited studies on the molecular biology of this behavior, however. We used an HIV-based lentivirus to produce germline transgenic zebra finches. The lentivirus en- lentivirus ͉ song system ͉ zebra finch

Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132–713) and STAT3 (127–722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition.

The high vocal center (HVC) of adult male canaries, Serinus canaria, is necessary for the production of learned song. New neurons are added to HVC every day, where they replace older neurons that have died, but the length of their survival depends on the time of year when they are born. A great number of HVC neurons born in the fall, when adult canaries learn a new song, are still present 8 mo later, when this song is used during the breeding season. By contrast, most of the neurons born in HVC in the spring, when little song learning takes place, disappear much sooner. Here we show that infusion of brain-derived neurotrophic factor into HVC during days 14 -20 after new HVC neurons are born in the spring confers on them a life expectancy comparable to that of fall-born neurons; this extension on life is not seen when infusion occurs 10 days earlier or later. We suggest that there is, in the adult HVC, a subset of neurons whose life expectancy is determined by brain-derived neurotrophic factor during a sensitive period soon after these neurons reach destination and start forming connections.N ew neurons are born in the adult canary brain in the ventricular zone lining the wall of the lateral ventricles (1, 2). They migrate into the high vocal center (HVC) and assume a sedentary phenotype 8-15 days after their birth. The number of these new neurons is much reduced during their third week of life (3). By day 30, the axons of many of the surviving new HVC neurons have reached their target, and many of the new cells have become a functional part of existing circuits (3-5). Thereafter, the life expectancy of the neurons depends on the time of year when they are born. Whereas the number of fall-born neurons is virtually the same 30 and 120 days after their birth, this number drops by one-half at the later survival time in spring-born neurons (6). However, the mechanism that regulates this season-dependent survival͞attrition has not been worked out in detail.In addition to season, the presence of new neurons in HVC has been shown to depend on variables such as amount of singing (7,8), auditory experience (9), blood testosterone levels (8, 10), and the presence of brain-derived neurotrophic factor (BDNF) in HVC (11). Some of these factors are related. For example, both amount of singing and blood testosterone levels change seasonally (12) and both influence the amount of BDNF present in HVC (8). There are at least two major sources of BDNF in HVC: neurons that project to the robust nucleus of the archistriatum (7) and endothelial cells lining HVC capillaries (13); in addition, a small percentage of HVC neurons that project to area X also express BDNF (7). Knowing that BDNF can influence neuron survival and that there is a dramatic decline in new neuron numbers 15-22 days after their birth (3), we decided to test whether BDNF infusion during this period could enhance the survival of new neurons born in the spring. We found that, whereas BDNF infusion into HVC during days 14-20 after new neurons are born markedly extends t...

Speech and vocal impairments characterize many neurological disorders. However, the neurogenetic mechanisms of these disorders are not well understood, and current animal models do not have the necessary circuitry to recapitulate vocal learning deficits. We developed germline transgenic songbirds, zebra finches (Taneiopygia guttata) expressing human mutant huntingtin (mHTT), a protein responsible for the progressive deterioration of motor and cognitive function in Huntington's disease (HD). Although generally healthy, the mutant songbirds had severe vocal disorders, including poor vocal imitation, stuttering, and progressive syntax and syllable degradation. Their song abnormalities were associated with HD-related neuropathology and dysfunction of the cortical-basal ganglia (CBG) song circuit. These transgenics are, to the best of our knowledge, the first experimentally created, functional mutant songbirds. Their progressive and quantifiable vocal disorder, combined with circuit dysfunction in the CBG song system, offers a model for genetic manipulation and the development of therapeutic strategies for CBG-related vocal and motor disorders.

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