Synergistic interactions between Cajal bodies and the miRNA processing machinery

Abstract

Our results demonstrate that Cajal bodies and the microRNA (miRNA) processing machinery functionally interact and together contribute to the biogenesis of miRNAs and small nuclear ribonucleoproteins.

Molecular determinants that govern scaRNA processing by Drosha/DGCR8

“…As shown in Fig. 4 B, processing assays using primary-scaRNA9 substrate incubated with FLAG beads complexed with FLAG-DGCR8/Drosha generate a fragment consistent with mgU2-30 (left panel, lanes 4 and 5), consistent with our previously published results ( Logan et al, 2020 ). A mgU2-30 fragment is not observed when using FLAG beads complexed with lysate from un-transfected cells (left panel, lane 3), or incubated with water alone (lane 2).…”

“…1 A, colored rectangles) are approximately the same size (70–80 nt) ( Tycowski et al, 2004 ) as precursor miRNAs generated by Drosha/DGCR8, indicating that some primary scaRNAs may be unorthodox substrates for the miRNA processing machinery. Our recent work supports this hypothesis ( Logan et al, 2020 ). To further investigate the mechanisms that generate scaRNA 2, 9 and 17 fragments, we have generated expression constructs in the pCDNA3.1 vector, which yield sufficient amounts of ectopically expressed full-length (FL) and fragment (frag) that can be easily detected by Northern blotting using specific DIG-labelled probes ( Fig.…”

“…Processing assays using FLAG beads from un-transfected cells (UT) and FLAG-DGCR8/Drosha transfected cells (Trans) were also conducted using primary-scaRNA2 (middle panel) and primary-scaRNA17 (right panel). As with primary scaRNA9 ( Logan et al, 2020 ) ( Fig. 4 B, left panel), fragments corresponding to mgU2-61 (middle panel) and mgU4-8 (right panel) are detected only in reactions using FLAG beads complexed with Drosha/DGCR8 (Trans) and not with FLAG beads from un-transected (UT) cells or incubated with water alone.…”

“…We have published that Drosha/DGCR8 complexes can process a primary-scaRNA9 substrate in vitro , generating both mgU2-19 and mgU2-30 fragments ( Logan et al, 2020 ). We have now examined if Drosha/DGCR8 can generate the mgU2-61 fragment using primary-scaRNA2 and the mgU4-8 fragment using primary-scaRNA17 in vitro transcribed substrates in an in vitro processing assay ( Fig.…”

“…We have recently published a paper demonstrating that Drosha and DGCR8, well-characterized components of the miRNA processing pathway ( Han et al, 2006 ; Macias et al, 2013 ; Nguyen et al, 2015 ), take part in the biogenesis of fragments derived from primary-scaRNA9 ( Logan et al, 2020 ). In addition, we have shown that CBs can associate with a miRNA gene cluster (the chromosome 19 microRNA cluster, C19MC) and positively regulate miRNA biogenesis ( Logan et al, 2020 ). These findings demonstrate a functional relationship between CBs and the miRNA processing machinery.…”