NOT2 Proteins Promote Polymerase II–Dependent Transcription and Interact with Multiple MicroRNA Biogenesis Factors in Arabidopsis    

Abstract

MicroRNAs (miRNAs) play key regulatory roles in numerous developmental and physiological processes in animals and plants. The elaborate mechanism of miRNA biogenesis involves transcription and multiple processing steps. Here, we report the identification of a pair of evolutionarily conserved NOT2_3_5 domain-containing-proteins, NOT2a and NOT2b (previously known as At-Negative on TATA less2 [NOT2] and VIRE2-INTERACTING PROTEIN2, respectively), as components involved in Arabidopsis thaliana miRNA biogenesis. NOT2 was identified by its interaction with the Piwi/Ago/Zwille domain of DICER-LIKE1 (DCL1), an interaction that is conserved between rice (Oryza sativa) and Arabidopsis thaliana. Inactivation of both NOT2 genes in Arabidopsis caused severe defects in male gametophytes, and weak lines show pleiotropic defects reminiscent of miRNA pathway mutants. Impairment of NOT2s decreases the accumulation of primary miRNAs and mature miRNAs and affects DCL1 but not HYPONASTIC LEAVES1 (HYL1) localization in vivo. In addition, NOT2b protein interacts with polymerase II and other miRNA processing factors, including two cap binding proteins, CBP80/ABH1, CBP20, and SERRATE (SE). Finally, we found that the mRNA levels of some protein coding genes were also affected. Therefore, these results suggest that NOT2 proteins act as general factors to promote the transcription of protein coding as well as miRNA genes and facilitate efficient DCL1 recruitment in miRNA biogenesis.

CDC5, a DNA binding protein, positively regulates posttranscriptional processing and/or transcription of primary microRNA transcripts

“…This was not unexpected, as CDC5 may be weakly associated with the DCL1 complex, or its association with HYL1 may need bridge proteins. In fact, NOT2 has been shown to interact with DCL1 and SE, but not HYL1 (8).…”

“…In Arabidopsis, pri-miRNAs are primarily transcribed by DNA-dependent RNA polymerase II (Pol II), with assistance from the mediator complex and the transcription factor Negative on TATA less2 (NOT2) (7,8). After transcription, pri-miRNAs are processed by an RNase III enzyme called DICER-LIKE1 (DCL1) to miRNA precursors and then to mature miRNAs (9, 10).…”

“…Four other proteins, DAWDLE (DDL; an RNA binding protein), Cap-binding protein 20, Cap-binding protein 80, and NOT2, which are associated with the DCL1 complex (8,(20)(21)(22), also function in miRNA biogenesis. Recent studies also reveal that the correct localization of DCL1 requires NOT2 and MODIFIER OF SNC1, 2 (an RNA binding protein) (8,23). In addition, the accumulation of a subset of miRNAs requires a proline-rich protein named SICKLE (24).…”

The crosstalk between plant microRNA biogenesis factors and the spliceosome

“…28 Other proteins required for proper plant miRNA biogenesis are DDL, STA1, SICLE (SIC), NOT2, and RACK1. [29][30][31][32][33] Mutations in the splicing factor STA1 gene have been shown to interfere with both premRNA and pri-miRNA splicing that leads to the accumulation of pri-miRNAs and concurrently to the downregulation of miRNA expression. 34 NOT2a and NOT2b proteins were suggested to participate in coupling the RNA Pol II transcriptional complex with the pri-miRNA processing machinery.…”

“…34 NOT2a and NOT2b proteins were suggested to participate in coupling the RNA Pol II transcriptional complex with the pri-miRNA processing machinery. 32 Thus, recent identification of these new components of the miRNA biogenesis pathway suggests that very complex set of processing events leading from miRNA primary transcripts to miRNA/miRNA* duplexes takes place in the nucleus, and that many aspects of pri-miRNA processing still require further studies to identify all players and interactions involved in this process.…”