hnRNP A1 Binds to the Terminal and Internal Loops of Pri-miR-18a, Causing Relaxation of the Stem
(A) Footprint analysis of the pri-miR-18a_73 nt/hnRNP A1 complex. Cleavage patterns were obtained for 5′ ³²P-labeled pri-miR-18a transcript (100 × 10³ c.p.m.) incubated in the presence of increasing concentrations of recombinant hnRNP A1 protein (+, 50 ng; ++, 100 ng; +++, 150 ng; ++++, 200 ng), treated with Pb (II)-lead ions (0.5 mM). F and T identify nucleotide residues subjected to partial digest with formamide (every nucleotide) or ribonuclease T1 (G-specific cleavage), respectively. Thick lines on the right-hand side indicate rows of nucleotides protected by hnRNP A1. Positions of selected residues are indicated.
(B) Cleavage pattern obtained for the unlabeled pri-miR-17-18a-19 RNA cluster incubated in the presence of hnRNP A1 protein (+ 50ng), treated with Pb (II)-lead ions (0.5 mM) and ribonuclease V1 (0.5 u/ml), detected by RT with a pri-miR-18a-specific ³²P-labeled oligonucleotide.
(C) Proposed structure of free and hnRNP A1-bound pri-miR-18a. The sites and intensities of cleavages generated by structure probes (presented below), located at the places of hnRNP A1 binding are shown. Nucleotides are numbered from the 5′ site of Drosha cleavage.

Multiple Sequence Alignments for the Human Genome with 16 Other Vertebrate Genomes Show Very High Level of Conservation in the Terminal Loop Regions of Some Human pri-miRNAs
(A) Conservation across pri-miR-18a corresponds to a persistently high phastCons conservation profile (the blue track labeled “Conservation”) across stem and terminal loop region predicted by miRBase and EvoFold.
(B) Conservation across pri-miR-27a includes a clear dip in phastCons conservation profile (the blue track labeled “Conservation”) corresponding to the terminal loop region predicted as above.
(C) A list of 74 pri-miRNAs with unusually highly conserved terminal loops is shown.

Truncated Stem Loops of Pri-miRNAs with Conserved Terminal Loops Bind Common and Distinct RNA-Binding Proteins
(A) RNA chromatography combined with Mass Spectrometry of selected pri-miRNAs harboring conserved loops was performed in HeLa nuclear extracts. Samples resulting from RNA chromatography were resolved on SDS gel and visualized using GelcodeBlue (Pierce). Bands 1 and 3 correspond to hnRNP A1, band 2 corresponds to hnRNP L, and band 4 corresponds to hnRNP I (PTB).
(B) A close-up of a region of the gel shown in (A). Expectation values from the mass spectrometry analysis are shown.
(C) Western blot analysis of RNA chromatography with truncated stem loops of corresponding pri-miRNAs.
(D) Predicted structures of pri-let-7a-1 and pri-miR-101-1 with highlighted putative protein binding sites are shown.

The Conserved Terminal Loops of Pri-miR-101-1 and Pri-let-7a-1 Are Required for Their Efficient Cleavage by Drosha
(A) Predicted secondary structures of wild-type and terminal loop mutants of pri-miR-101-1 (pri-miR-101-1_loop_mt1 and pri-miR-101-1_loop_mt2). Mutated nucleotides are bolded.
(B) In vitro processing of wild-type pri-miR-101-1 and loop mutants. Radiolabeled pri-RNAs (50 × 10³ c.p.m.) were incubated in HeLa cell extracts (lanes 2, 4, and 6). Lanes 1, 3, and 5 show negative controls with no extract added. Products were analyzed on an 8% polycrylamide gel. M, RNA size marker.
(C) Predicted secondary structures of wild-type and terminal loop mutants of pri-let-7a-1.
(D) In vitro processing of pri-let-7a-1 and loop mutants. Radiolabeled pri-RNAs (50 × 10³ c.p.m.) were incubated in HeLa cell extracts (lanes 2, 4, and 6). Lanes 1, 3, and 5 show negative controls with no extract added. Products were analyzed on an 8% polycrylamide gel. M, RNA size marker.

A Fine Structural Alteration in the Pri-miR-18a Stem Allows for hnRNP A1-Independent Drosha Cleavage
(A) Sequences of the highly related pri-miR-18a and pri-miR-18b.
(B) Schematic of the secondary structures of wild-type and stem mutants of pri-miR-18a and pri-miR-18b. Mutated nucleotides are bolded.
(C) In vitro processing of pri-miR-18a and the mutant pri-miR-18a UC > GU substrate. Both radiolabeled primary RNA sequences (50 × 10³ c.p.m.) were incubated in the presence of either control HeLa extracts (lanes 2 and 5) or hnRNP A1-depleted extracts (lanes 3 and 6). Lanes 1 and 4 show negative controls with no extract added. Products were analyzed on an 8% polycrylamide gel. The asterisk indicates the pre-miR-18a product. M, RNA size marker.
(D) In vitro processing of pri-miR-18b and the mutant pri-miR-18b GU > UC substrate. Both radiolabeled primary RNA sequences (50 × 10³ c.p.m.) were incubated in control HeLa extracts (lanes 2 and 4). Lanes 1 and 3 show negative controls with no extract added. Products were analyzed on an 8% polycrylamide gel. The asterisk indicates the pre-miR-18b product. M, RNA size marker.

LooptomiRs Designed against Conserved Terminal Loops Block the Processing of a Subset of Pri-miRNAs
(A) Primary RNA transcripts corresponding to pri-miR-16-1 (lanes 1–5) and pri-miR-18a (lanes 6–10) (50 × 10³ c.p.m.) were processed in HeLa extracts in the presence of specific (lanes 3 and 4 and 8 and 9 for pri-miR-16-1 and pri-miR-18a, respectively) or control (lane 5 for miR-16-1 and lane 10 for miR-18a, respectively) looptomiRs (+, 50 μM; ++, 100 μM). Lanes 1 and 6 show negative controls with no extract added. M, RNA size marker.
(B) Processing of pri-miR-27a is not affected by the addition of a specific looptomiR. The RNA substrate (50 × 10³ c.p.m.) was processed in vitro in the presence of the corresponding blocking looptomiR (lane 3) or a control looptomiR (lane 4).
(C and D) In vitro processing of pri-miRNAs 101-1 and let-7a-1 is sensitive to the presence of specific looptomiRs. Radiolabeled pri-miRNA 101-1 and let-7a-1, (50 × 10³ c.p.m.) were processed in HeLa extracts with the addition of a specific looptomiR (lane 3) or with a control looptomiR specific for miR-18a (lane 4). As a control, the reaction was also carried out in the absence of any looptomiR (lanes 2). Lanes 1 represent control reaction without extract. M, RNA size marker.

The Conserved Terminal Loop of pri-miR-18a Is Required for Its Efficient Cleavage by Drosha
(A) Validated secondary structures of wild-type and terminal loop mutants of pri-miR-18a (pri-miR-18a_loop_mt1 and pri-miR-18a_loop_mt2). Mutated nucleotides are bolded.
(B) In vitro processing of wild-type pri-miR-18a and loop mutants. Radiolabeled pri-RNAs (50 × 10³ c.p.m.) were incubated in HeLa cell extracts (lanes 2, 4, and 6). Lanes 1, 3, and 5 show negative controls with no extract added. Products were analyzed on an 8% polycrylamide gel. The asterisk indicates the single-stranded RNA regions after Drosha cleavage. M, RNA size marker.
(C) Predicted secondary structures of wild-type and terminal loop mutants of pri-miR-16-1.
(D) In vitro processing of pri-miR-16-1 and loop mutants pri-miR-16-1_loop_mt1 and pri-miR-16-1_loop_mt2. Radiolabeled pri-RNAs (50 × 10³ c.p.m.) were incubated in HeLa cell extracts (lanes 2, 4, and 6). Lanes 1, 3, and 5 show negative controls with no extract added. Products were analyzed on an 8% polycrylamide gel. The asterisk indicates the single-stranded RNA regions after Drosha cleavage. M, RNA size marker.