MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

MicroRNAs are a family of small, non-coding RNAs that regulate gene expression in a sequence-specific manner. The two founding members of the microRNA family were originally identified in Caenorhabditis elegans as genes that were required for the timed regulation of developmental events. Since then, hundreds of microRNAs have been identified in almost all metazoan genomes, including worms, flies, plants and mammals. MicroRNAs have diverse expression patterns and might regulate various developmental and physiological processes. Their discovery adds a new dimension to our understanding of complex gene regulatory networks.

/pdf/micrornas-small-rnas-with-a-big-role-in-gene-regulation-3a05xihg95.pdf

MicroRNAs: small RNAs with a big role in gene regulation

RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

/pdf/role-for-a-bidentate-ribonuclease-in-the-initiation-step-of-4ptw8d0dcg.pdf

Role for a bidentate ribonuclease in the initiation step of RNA interference

In Caenorhabditis elegans, lin-4 and let-7 encode 22- and 21-nucleotide (nt) RNAs, respectively, which function as key regulators of developmental timing. Because the appearance of these short RNAs is regulated during development, they are also referred to as small temporal RNAs (stRNAs). We show that many 21- and 22-nt expressed RNAs, termed microRNAs, exist in invertebrates and vertebrates and that some of these novel RNAs, similar to let-7 stRNA, are highly conserved. This suggests that sequence-specific, posttranscriptional regulatory mechanisms mediated by small RNAs are more general than previously appreciated.

/pdf/identification-of-novel-genes-coding-for-small-expressed-4c05eip7o1.pdf

Identification of novel genes coding for small expressed RNAs.

MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and pathological processes in animals. The biogenesis of miRNAs is under tight temporal and spatial control, and their dysregulation is associated with many human diseases, particularly cancer. In animals, miRNAs are ∼22 nucleotides in length, and they are produced by two RNase III proteins--Drosha and Dicer. miRNA biogenesis is regulated at multiple levels, including at the level of miRNA transcription; its processing by Drosha and Dicer in the nucleus and cytoplasm, respectively; its modification by RNA editing, RNA methylation, uridylation and adenylation; Argonaute loading; and RNA decay. Non-canonical pathways for miRNA biogenesis, including those that are independent of Drosha or Dicer, are also emerging.

Regulation of microRNA biogenesis

https://www.umassmed.edu/globalassets/mellolab/documents/publications/tabara_rde1_rnai_screen_1999.pdf

The rde-1 Gene, RNA Interference, and Transposon Silencing in C. elegans

In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues. We have named this product nucampholin. Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes. Nucampholin resides in all nuclei of C. elegans and is essential in early development and in differentiating tissue. Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression. Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline. The broad requirements allowed investigation of soma-germline differences in gene expression. When introduced into standard transgene arrays, let-858 (like many other C. elegans genes) functions well in soma but poorly in germline. We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like. To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA. The resulting transgenes show robust expression in both germline and soma. Our results suggest the possibility of concerted mechanisms for silencing unwanted germiline expression of repetitive sequences.

https://www.genetics.org/content/genetics/146/1/227.full.pdf

Distinct Requirements for Somatic and Germline Expression of a Generally Expressed Caernorhabditis elegans Gene

RNA polymerase II is a glycoprotein : modification of the cooh-terminal domain by O-GlcNAc

PERSPECTIVES AND SUMMARY 841 BINDING STUDIES USING LECTINS .. .. "........ 843 Lectin Binding Sites in the Nucleus"""""""""" . . """ . . " . . . . " . . " . . """" . . . . . . . 843 Lectin Binding Sites in the Cytoplasm 848 BIOCHEMICAL STUDIES ... . . .. . . . . . . . .. . .. . 849 General Compositional Analyses and Metabolic Labeling Studies ... ... """""...... 849 High Mobility Group Glycoproteins"""""""""""" . ......... """""""""" . . . . . 851 Proteoglycans and Glycosaminoglycans 851 Glycogenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 857 Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 857 O-Linked N-Acetylglucosamine-Bearing Glycoproteins 858 NUCLEAR AND CYTOPLASMIC SACCHARIDE-BINDING PROTEINS""""""" 863 Endogenous Lectins . . . . . . .. . . .. . . . . . . . . . . ... .. . ...... . . . ..... 863 Glycosidases . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 866 Glycosyltransferases ...... ... . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 867 FUTURE DIRECTIONS . . . . 868

Glycosylation in the nucleus and cytoplasm

Germline maintenance in the nematode C. elegans requires global repressive mechanisms that involve chromatin organization. During meiosis, the X chromosome in both sexes exhibits a striking reduction of histone modifications that correlate with transcriptional activation when compared with the genome as a whole. The histone modification spectrum on the X chromosome corresponds with a lack of transcriptional competence, as measured by reporter transgene arrays. The X chromosome in XO males is structurally analogous to the sex body in mammals, contains a histone modification associated with heterochromatin in other species and is inactivated throughout meiosis. The synapsed X chromosomes in hermaphrodites also appear to be silenced in early meiosis, but genes on the X chromosome are detectably expressed at later stages of oocyte meiosis. Silencing of the sex chromosome during early meiosis is a conserved feature throughout the nematode phylum, and is not limited to hermaphroditic species.

William G. Kelly

Papers

The rde-1 Gene, RNA Interference, and Transposon Silencing in C. elegans

Distinct Requirements for Somatic and Germline Expression of a Generally Expressed Caernorhabditis elegans Gene

RNA polymerase II is a glycoprotein : modification of the cooh-terminal domain by O-GlcNAc

Glycosylation in the nucleus and cytoplasm

X-chromosome silencing in the germline of C. elegans.