Summary The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor (Figure 1A,B [1,2]) is a heterotrichous ciliate distantly related to familiar ciliate models such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 millimeter long. Early developmental biologists, including T.H. Morgan[3], were attracted to the system because of its regenerative abilities -- if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality [2,3]. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms [4]. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome: First, we find that Stentor uses the standard genetic code, suggesting that ciliate specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor’s cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor.

During nuclear maturation of most eukaryotic pre-messenger RNAs and long non-coding RNAs, introns are removed through the process of RNA splicing. Different classes of introns are excised by the U2-type or the U12-type spliceosomes, large complexes of small nuclear ribonucleoprotein particles and associated proteins. We created intronIC, a program for assigning intron class to all introns in a given genome, and used it on 24 eukaryotic genomes to create the Intron Annotation and Orthology Database (IAOD). We then used the data in the IAOD to revisit several hypotheses concerning the evolution of the two classes of spliceosomal introns, finding support for the class conversion model explaining the low abundance of U12-type introns in modern genomes.

Expansion and transformation of the minor spliceosomal system in the slime mold Physarum polycephalum Highlights d >20,000 minor introns in P. polycephalum, 25 times more than any other species d Co-evolution of the minor spliceosome and minor intron splicing signals d Thousands of introns with non-canonical splice sites d Highly efficient splicing of minor introns, in contrast to other species

Background Two spliceosomal intron types co-exist in eukaryotic precursor mRNAs and are excised by distinct U2-dependent and U12-dependent spliceosomes. In the diplomonad Giardia lamblia , small nuclear (sn) RNAs show hybrid characteristics of U2- and U12-dependent spliceosomal snRNAs and 5 of 11 identified remaining spliceosomal introns are trans -spliced. It is unknown whether unusual intron and spliceosome features are conserved in other diplomonads. Results We have identified spliceosomal introns, snRNAs and proteins from two additional diplomonads for which genome information is currently available, Spironucleus vortens and Spironucleus salmonicida , as well as relatives, including 6 verified cis- spliceosomal introns in S. vortens . Intron splicing signals are mostly conserved between the Spironucleus species and G. lamblia . Similar to ‘long’ G. lamblia introns, RNA secondary structural potential is evident for ‘long’ (> 50 nt) Spironucleus introns as well as introns identified in the parabasalid Trichomonas vaginalis . Base pairing within these introns is predicted to constrain spatial distances between splice junctions to similar distances seen in the shorter and uniformly-sized introns in these organisms. We find that several remaining Spironucleus spliceosomal introns are ancient. We identified a candidate U2 snRNA from S. vortens , and U2 and U5 snRNAs in S. salmonicida ; cumulatively, illustrating significant snRNA differences within some diplomonads. Finally, we studied spliceosomal protein complements and find protein sets in Giardia , Spironucleus and Trepomonas sp. PC1 highly- reduced but well conserved across the clade, with between 44 and 62 out of 174 studied spliceosomal proteins detectable. Comparison with more distant relatives revealed a highly nested pattern, with the more intron-rich fornicate Kipferlia bialata retaining 87 total proteins including nearly all those observed in the diplomonad representatives, and the oxymonad Monocercomonoides retaining 115 total proteins including nearly all those observed in K. bialata . Conclusions Comparisons in diplomonad representatives and species of other closely-related metamonad groups indicates similar patterns of intron structural conservation and spliceosomal protein composition but significant divergence of snRNA structure in genomically-reduced species. Relative to other eukaryotes, loss of evolutionarily-conserved snRNA domains and co...

While the vast majority of spliceosomal introns are removed by the so-called U2 (major) spliceosome, diverse eukaryotes also contain a mysterious second form, the U12 (minor) form, and associated introns. In all characterized species, U12-type introns are distinguished by several features, including being rare in the genome, containing extended evolutionary-conserved splicing sites, being generally ancient as judged by conservation between distant species, and being inefficiently spliced. Here, we report a remarkable exception in the slime mold Physarum polycephalum. The P. polycephalum genome contains > 20,000 U12-type introns -- 25 times more than any other species -- with transformed splicing signals that have co-evolved with the spliceosome due to massive gain of efficiently spliced U12-type introns. These results reveal an unappreciated dynamism of minor spliceosomal introns and spliceosomal introns in general.