The RNA splicing endonuclease cleaves two phosphodiester bonds within folded precursor RNAs during intron removal, producing the functional RNAs required for protein synthesis. Here we describe at a resolution of 2.85 angstroms the structure of a splicing endonuclease from Archaeglobus fulgidus bound with a bulge-helix-bulge RNA containing a noncleaved and a cleaved splice site. The endonuclease dimer cooperatively recognized a flipped-out bulge base and stabilizes sharply bent bulge backbones that are poised for an in-line RNA cleavage reaction. Cooperativity arises because an arginine pair from one catalytic domain sandwiches a nucleobase within the bulge cleaved by the other catalytic domain.

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Histone deacetylases (HDACs) are part of a system that links epigenetic control of gene expression to a variety of environmental stimuli. Some HDACs, including HDAC4, shuttle between the cytoplasm and nucleus in response to physiological cues such as calcium signaling. HDAC4 mRNA is enriched in the brain, but the regional and subcellular protein expression pattern of HDAC4 is not known. Here we show that HDAC4 is more highly expressed in some brain regions than in others. HDAC4 is present in the perikaryial cytoplasm of most neurons but its nuclear localization is variable. In some areas, such as the dentate gyrus, nuclear expression is not detectable, whereas in other areas some neuronal nuclei contain HDAC4 immunoreactivity whereas others do not. In the cytoplasm, HDAC4 immunoreactivity is punctate. Some of these puncta are present in dendritic spines where the strongest immunoreactivity is associated with the postsynaptic density. These data demonstrate that the regional and subcellular distribution of HDAC4 is heterogeneous and raise the possibilities that HDAC4 acts on nonhistone substrates in dendritic spines or that it shuttles between spine and nucleus to coordinate synaptic activity with gene expression.

The RNA-splicing endonuclease is an evolutionarily conserved enzyme responsible for the excision of introns from nuclear transfer RNA (tRNA) and all archaeal RNAs. Since its first identification from yeast in the late 1970s, significant progress has been made toward understanding the biochemical mechanisms of this enzyme. Four families of the splicing endonucleases possessing the same active sites and overall architecture but with different subunit compositions have been identified. Two related consensus structures of the precursor RNA splice sites and the critical elements required for intron excision have been established. More recently, a glimpse was obtained of the structural mechanism by which the endonuclease recognizes the consensus RNA structures and cleaves at the splice sites. This review summarizes these findings and discusses their implications in the evolution of intron removal processes.

Among the tRNA population of the archaeal parasite Nanoarchaeum equitans are five species assembled from separate 5 and 3 tRNA halves and four species derived from tRNA precursors containing introns. In both groups an intervening sequence element must be removed during tRNA maturation. A bulge-helixbulge (BHB) motif is the hallmark structure required by the archaeal splicing endonuclease for recognition and excision of all introns. BHB motifs are recognizable at the joining sites of all five noncontinuous tRNA species, although deviations from the canonical BHB motif are clearly present in at least two of them. Here, we show that the N. equitans splicing endonuclease cleaves tRNA precursors containing normal introns, as well as all five noncontinuous precursor tRNAs, at the predicted splice sites, indicating the enzyme's dual role in the removal of tRNA introns and processing of tRNA halves to be joined in trans. The cleavage activity on a set of synthetic canonical and noncanonical BHB constructs showed that the N. equitans splicing endonuclease accepts a broader range of substrates than the homodimeric Archaeoglobus fulgidus enzyme. In contrast to the A. fulgidus endonuclease, the N. equitans splicing enzyme possesses two different subunits. This heteromeric endonuclease type, found in N. equitans, in all Crenarchaeota, and in Methanopyrus kandleri, is able to act on the noncanonical tRNA introns present only in these organisms, which suggests coevolution of enzyme and substrate. tRNA processing M any tRNA genes in Eukarya and Archaea contain intron sequences that must be removed during tRNA processing to generate mature, functional tRNAs. The first step of tRNA splicing is catalyzed by a specific endonuclease that excises the intron from the precursor tRNA (pre-tRNA), yielding two tRNA half-molecules, a 5Ј half-exon ending with a 2Ј,3Ј-cyclic phosphate, and a 3Ј half-exon with a 5Ј-hydroxyl terminus (1, 2). This step is followed by the joining of the tRNA exons catalyzed by a tRNA splicing-specific ligase in Eukarya; however, little is known about the tRNA ligation mechanism in Archaea (3). The archaeal splicing endonuclease recognizes a consensus bulgehelix-bulge (BHB) structure that is found at the intron-exon junctions of intron-containing tRNA (4), rRNA (5, 6), and mRNA (7). The BHB motif exhibits pseudo-twofold symmetry with the two 3-nt bulges present on the same minor groove face of the central 4-bp helix, termed the 3-4-3 BHB (8). This structure makes the splice sites well positioned for attack by the splicing endonuclease. Two archaeal endonuclease families are found in Euryarchaeota: (i) ␣ 2 homodimeric enzymes of 288-353 aa in length or (ii) ␣ 4 homotetrameric enzymes of Ͻ200 aa in length (1, 9). These endonucleases seem to have a strict requirement for the pseudosymmetric 3-4-3 BHB RNA (4). Recently, a third family of splicing endonucleases was discovered in the Crenarchaeon Sulfolobus solfataricus; the enzyme has a heteromeric subunit composition (10). Multiple sequence alignments and BLAST searc...