Invasion by exogenous RNA:cellular defense strategies and implications for RNA inference

Exogenous RNA poses a continuous threat to genome stability and integrity across various organisms.Accumulating evidence reveals complex mechanisms underlying the cellular response to exogenous RNA,including endo-lysosomal degradation,RNA-dependent repression and innate immune clearance.Across a variety of mechanisms,the natural anti-sense RNAdependent defensive strategy has been utilized both as a powerful gene manipulation tool and gene therapy strategy named RNA-interference(RNAi).To optimize the efficiency of RNAi silencing,a comprehensive understanding of the whole life cycle of exogenous RNA,from cellular entry to its decay,is vital.In this paper,we review recent progress in comprehending the recognition and elimination of foreign RNA by cells,focusing on cellular entrance,intracellular transportation,and immune-inflammatory responses.By leveraging these insights,we highlight the potential implications of these insights for advancing RNA interference efficiency,underscore the need for future studies to elucidate the pathways and fates of various exogenous RNA forms,and provide foundational information for more efficient RNA delivery methods in both genetic manipulation and therapy in different organisms.

Further analyses of variation of ribosome DNA copy number and polymorphism in ciliates provide insights relevant to studies of both molecular ecology and phylogeny

Sequence-based approaches, such as analyses of ribosome DNA(rDNA) clone libraries and high-throughput amplicon sequencing, have been used extensively to infer evolutionary relationships and elucidate the biodiversity in microbial communities.However, recent studies demonstrate both r DNA copy number variation and intra-individual(intra-genomic) sequence variation in many organisms, which challenges the application of the rDNA-based surveys. In ciliates, an ecologically important clade of microbial eukaryotes, rDNA copy number and sequence variation are rarely studied. In the present study, we estimate the intraindividual small subunit rDNA(SSU r DNA) copy number and sequence variation in a wide range of taxa covering nine classes and 18 orders of the phylum Ciliophora. Our studies reveal that:(i) intra-individual sequence variation of SSU rDNA is ubiquitous in all groups of ciliates detected and the polymorphic level varies among taxa;(ii) there is a most common version of SSU rDNA sequence in each cell that is highly predominant and may represent the germline micronuclear template;(iii)compared with the most common version, other variant sequences differ in only 1–3 nucleotides, likely generated during macronuclear(somatic) amplification;(iv) the intra-cell sequence variation is unlikely to impact phylogenetic analyses;(v) the rDNA copy number in ciliates is highly variable, ranging from 103 to 106, with the highest record in Stentor roeselii. Overall,these analyses indicate the need for careful consideration of SSU r DNAvariation in analyses of the role of ciliates in ecosystems.

A paradox： rapid evolution rates of germline-limited sequences are associated with conserved patterns of rearrangements in cryptic species of Chilodonella uncinata(Protista, Ciliophora)

Some of the most extreme genome wide rearrangements are found in ciliates, which are unique in possessing both germline micronucleus and somatic macronucleus in every cell/organism. A series of DNA rearrangement events, including DNA elimination, chromosomal fragmentation, gene unscrambling and alternative processing, happen during macronuclear development. To assess the molecular evolution of macronuclear and germline-limited sequences in different cryptic species of Chilodonella uncinata, we characterized the actin, α-tubulin and β-tubulin genes in the micronucleus and macronucleus genomes of USA-SC2 strain and compared them with other strains(i.e. cryptic species). Three main results are:(i) rearrangement patterns between germline and soma are conserved for each gene among the cryptic species of C. uncinata;(ii) in contrast, the germlinelimited regions are highly divergent in sequence and length among the cryptic species;(iii) pointer shifting is frequent among the cryptic species. We speculate that pointer sequences may serve as the buffer between the conserved macronuclear destined sequences and rapidly-evolving internal eliminated sequences. The data combined with previous studies demonstrate the plasticity of gene rearrangement among different groups of ciliates and add to the growing data for the role of genome rearrangements in species differentiation.

Timing and characteristics of nuclear events during conjugation and genomic exclusion in Paramecium multimicronucleatum

Ciliated protists are ideal material for studying the origin and evolution of sex,because of their nuclear dimorphism(containing both germline micronucleus and somatic macronucleus in the same cytoplasm),special sexual processes(conjugation and autogamy),and high diversity of mating-type systems.However,the study of sexual process is limited to only a few species,due to the difficulties in inducing or observing conjugation.In the present study,we investigate the conjugation process in Paramecium multimicronucleatum:(1)of the three prezygotic divisions,all micronuclei undergo the first two divisions(meiosis I,II),while a variable number of nuclei undergo the third division(mitosis);(2)the synkaryon divides three times after fertilization,giving rise to eight products that differentiate into four macronuclear anlagen and four micronuclei;(3)cells restore the vegetative stage after two successive cell fissions during which the macronuclear anlagen are distributed into daughter cells without division,while micronuclei divide mitotically;(4)the parental macronucleus begins to fragment following the first meiotic division and finally degenerates completely;(5)the entire process takes about 110 h,of which about 85 h are required for macronuclear development.In addition,we describe for the first time the process of genomic exclusion occurring between amicronucleate and micronucleate cells of P.multimicronucleatum,during which the micronucleate cell contributes a pronucleus to the amicronucleate cell,resulting in both exconjugants being homozygotes.These results provide new insights into the diversity of sexual processes and lay an important cytological basis for future in-depth studies of mating systems in ciliates.

New contributions to the phylogeny of the ciliate class Heterotrichea(Protista,Ciliophora):analyses at family-genus level and new evolutionary hypotheses

Heterotrichous ciliates play an important role in aquatic ecosystem energy flow processes and many are model organisms for research in cytology,regenerative biology,and toxicology.In the present study,we combine both morphological and molecular data to infer phylogenetic relationships at family-genus level and propose new evolutionary hypotheses for the class Heterotrichea.The main results include:(1)96 new ribosomal DNA sequences from 36 populations,representing eight families and 13 genera,including three poorly annotated genera,Folliculinopsis,Ampullofolliculina and Linostomella;(2)the earliest-branching families are Spirostomidae in single-gene trees and Peritromidae in the concatenated tree,but the family Peritromidae probably represents the basal lineage based on its possession of many"primitive"morphological characters;(3)some findings in molecular trees are not supported by morphological evidence,such as the family Blepharismidae is one of the most recent branches and the relationship between Fabreidae and Folliculinidae is very close;(4)the systematic positions of Condylostomatidae,Climacostomidae,and Gruberiidae remain uncertain based either on morphological or molecular data;and(5)the monophyly of each genus included in the present study is supported by the molecular phylogenetic trees,except for Blepharisma in the SSU r DNA tree and Folliculina in the ITS1-5.8 S-ITS2 tree.

Restoring Vision Naturally and Noninvasively

More than 200 million people across the world suffer from moderate to severe visual impairments,mostly due to retinal degeneration or damage to the eyes[1].Methods of reprogramming the cells in the eyes or electrical stimulation can be used to restore vision in acquired blindness[2].

New considerations on the phylogeny of Sessilida(Protista:Ciliophora:Peritrichia)based on multiple-gene information,with emphasis on colonial taxa

The subclass Peritrichia,containing two orders Sessilida and Mobilida,is a major group of ciliates with worldwide distribution and high species diversity.Several studies have investigated the phylogeny of peritrichs;however,the evolutionary relationships and classification of some families and genera within the Sessilida remain unclear.In the present study,we isolated and identified 22 peritrich populations representing four families and six genera and obtained 64 rDNA sequences to perform phylogenetic analyses and assess their systematic relationships.Ancestral character reconstruction was also carried out to infer evolutionary routes within the Sessilida.The results indicate:(1)family Vaginicolidae is monophyletic and acquisition of the typical peritrich lorica represents a single evolutionary divergence;(2)core epistylidids evolved from a Zoothamnium-like ancestor and experienced spasmoneme loss during evolution;(3)Campanella clusters with species in the basal clade and shows stable morphological differences with other epistylidids,supporting its assignment to a separate family;(4)the structure of the peristomial lip may be a genus-level character rather than a diagnostic character for discriminating Epistylididae and Operculariidae,thus a redefinition of Operculariidae should be carried out when more species have been investigated;(5)some characters,such as lifestyle(solitary or colonial),spasmoneme and living habit(sessile or free-swimming),evolved repeatedly among sessilids indicating that species with non-contractile stalks or that are free-swimming have multiple evolutionary routes and might derive from any sessilid lineage without a lorica.The close phylogenetic relationships of some morphologically distinct sessilids imply that the diagnoses of some genera and families should be improved.