Major contribution of transcription initiation to 5’-end formation of mitochondrial steady-state transcripts in maize

Figures & data

Table 1. 5′ and 3′ termini of the steady-state transcripts in maize NB mitochondria.

Steady-state transcripts^1	Position of 5′ termini^2	Position of 3′ termini^3	Type of 5′ termini	RNA secondary structures nearby processed 5′ ends^4	RNA secondary structures or sRNAs at 3′ ends^5
nad1	−1,217~−1,211	+80~+82	Processed	t-element upstream	SLS upstream
nad2-1	−1,985	+34/+35 +34/+35 +34/+35 +34/+35 +34/+35	Primary		SLS downstream and sRNA
nad2-2	−1,557		Primary
nad2-3	−579~−552		Processed
nad2-4	−394~−377		Processed
nad2-5	−360~−56		Processed
nad3-rps12^6	−76~−73	+12~+26	Processed	tRNA^Leu upstream
nad4-1	−345	+29~+31 +29~+31	Primary		SLS downstream and sRNA
nad4-2	−241~−215		Processed
nad4L	−356~−197	+67	Primary
nad5-1^7	−374	+89~+92 +89~+92	Primary		tRNA^Cys downstream and sRNA
nad5-2	−308~−53		Processed
nad6-1	−498~−254	+681 +681	Primary		SLSs upstream and downstream
nad6-2	−227/-226		Processed
nad7	−322	+50/+51	Primary		sRNA
nad9-1	−811~−808	+57/+58 +57/+58	Primary		sRNA
nad9-2	−565~−527		Primary
cob	−874~−867	+114/+115	Primary		SLS downstream
cox1-1	−153	+466/+467 or +473/+475	Primary		SLS upstream of +466/+467 3′ end
cox1-2	−57		Processed
cox2-1^8	−1,701/−1,700 −1,701/−1,700	+360/+361	Primary		SLS upstream of the +39 3′ end
cox2-2		+39 +39 +39	Primary
cox2-3	−1,283~−1,276		Primary
cox2-4	−1,030~−992		Primary
cox3	−362~−315	+56/+57	Primary		SLS downstream
ccmB	−216~−214	+202~+204	Primary		SLS upstream
ccmC-1	−1,826	+34~+36 +34~+36 +34~+36 +34~+36	Primary		tRNA^Leu downstream
ccmC-2	−1,120		Primary
ccmC-3	−866/-865		Primary
ccmC-4	−456~−452		Processed
ccmFC	−798~−766	+34/+35	Primary
ccmFN-rps1-1 ^9	−2,136~−2,119	+72/+73 +72/+73	Primary
ccmFN-rps1-2	−1,109		Primary
atp1-1	−2,364~−2,361	+444/+445 +444/+445 +444/+445 +444/+445 +444/+445	Primary
atp1-2	−1,313~−1,307		Processed
atp1-3	−542~−319		Primary
atp1-4	−169~−108		Primary
atp1-5	+89/+90		Processed
atp4-1	−1,821~−1,794	+727~+732 +727~+732	Primary
atp4-2	−285~−278 −285~−278		Primary
atp4-3		+204~+206	Primary
atp6-1	−2,223/-2,221	+97/+98 +97/+98 +97/+98	Primary
atp6-2	−440		Primary
atp6-3	−350		Processed
atp6-nad6-1^10	−2,223/-2,221	+681 +681	Primary		SLSs upstream and downstream
atp6-nad6-2	−440		Primary
atp8-1	−384~−356	+104/+105 or +134/+135	Primary		Both +104/+105 and +134/+135 ends downstream of SLS
atp8-2	−134~−126		Processed
atp9-1^11	−635	+122 +122 +122	Primary		SLS upstream^12
atp9-2	−420~−377		Primary
atp9-3	−275~−268		Processed
rps2A	−995	+133~+135	Primary		SLS upstream
rps2B^13	−57	+517~+520	Processed
rps3-rpl16-1^14	−379~−373	+244/+245 +244/+245	Primary		SLS downstream
rps3-rpl16-2	−236		Processed	SLS downstream
rps4-1	−375 −375	+1826/+1834	Primary
rps4-2		+690	Primary
rps7	−319~−264	+117/+118	Primary
rps12ct-rps7ct^15	−673	+309/+310	Processed
rps13-1^15	−566/-565	+1,260~+1,271 +1,260~+1,271	Processed	tRNA^Met upstream
rps13-2	−291 −291		Processed	t-element upstream
rps13-3		+1,033~+1,048	Processed
rpl16-1	+27	+244/+245	Primary		SLS downstream
mttB	−189	+52~+54	Primary		SLS upstream
5S rRNA^16	419,506 ~ 419,509	419,386 ~ 419,388	Processed
18S rRNA^17	421,587 ~ 421,589	419,619 ~ 419,621	Processed
26S rRNA^18	400,232 ~ 400,236	403,744 ~ 403,748	Processed
nad1T1	−1,217~−1,211	+1,546/+1,547	Processed	t-element upstream
nad1T3	−758~755	+681/+682	Primary		SLS upstream
nad1T4	−237~ −74	+80~+82	Processed		SLS upstream
nad2T1-1	−1,985	+683~+685 +683~+685	Primary		SLS upstream
nad2T1-2	−1,557		Primary
nad2T2	−336~−330	+34~+36	Processed	tRNA^Met upstream	SLS downstream and sRNA
nad5T1-1^20	−374	+729~+733 +729~+733	Primary		SLS upstream
nad5T1-2	−334~−42		Processed
nad5T2	−154~−23	+641/+642	Processed		SLS downstream
nad5T3	−478~−32	+89~+92	Processed		tRNA^Cys downstream and sRNA

Footnote: ¹Many mature and precursor RNAs are accumulated as multiple isoforms, and they are numbered sequentially. ²Positions of 5′ ends in respect to AUG (+1, for mature RNAs) or the neighboring downstream exon (+1, for precursor RNAs). ³Positions of 3′ termini in respect to translation stop codon UAA, UAG, or UGA (-1, for mature RNAs) or the neighboring upstream exon (-1, for precursor RNAs). ⁴RNA secondary structures (including SLSs, t-elements and tRNAs) directly upstream or downstream of the processed 5′ termini. ⁵RNA secondary structures (including SLSs, t-elements and tRNAs) adjacent to the mapped 3′ ends, and conservation of Arabidopsis mitochondrial sRNAs in maize. ⁶The 5′ and 3′ ends in respect to nad3 AUG (+1) and rps12 UGA (-1), respectively. ⁷The 5′ and 3′ termini of nad5 are identical with previously mapping results (Zhang et al., 2017). ⁸Identical with previously identified 3′ terminus (Williams et al., 2000). ⁹The 5′ and 3′ ends in respect to ccmFN AUG (+1) and rps1 UAA (-1), respectively. ¹⁰The 5′ and 3′ ends in respect to atp6 AUG (+1) and nad6 UAA (-1), respectively. ¹¹The 5′ and 3′ termini of atp9 are identical with previous results (Mulligan et al., 1988). ¹²A conserved SLS is present at the 3′ end of pea atp9, where it defines the transcript end by blocking 3′→5′ exonucleolytic processing (Dombrowski et al., 1997). ¹³In mapping of rps2B monocistron by cRT-PCR, the 5S rRNA-rps2B dicistron was always amplified. However, RNA gel blots using either rps2B or 5S rRNA probes could not detect the accumulation of 5S rRNA-rps2B discistron. ¹⁴The 5′ and 3′ ends in respect to rps3 AUG (+1) and rpl16 UAA (-1), respectively. ¹⁵The 5′ and 3′ ends in respect to rps12ct AUG (+1) and rps7ct UAA (-1), respectively. ¹⁶The 5′ termini of rps13-1 to -3 are likely generated by post-transcriptional processing since they are exactly downstream of a tRNA or t-element. ^{17, 18} and ¹⁹Positions of 5S, 18S and 26S rRNA termini in the NB mitochondrial genome, respectively. The mature 5S rRNA was rather small (~120 nts) that it was difficult to amplify the circularized 5S rRNA molecules by cRT-PCR, and its 5′ and 3′termini were determined by amplification of 26S-5S and 5S-26S fusion transcripts. The 5′ end of 26S rRNA and 3′ terminus of 18S rRNA are identical with previous results (Maloney et al., 1989; Williams et al., 2000). ²⁰The 5′ and 3′ termini of nad5-T1 are identical with previously mapping results (Zhang et al., 2017).

Figure 1. Mapping of representative mature RNAs with primary 5′ ends. (A) Gel separation of cRT-PCR products. + and -: samples treated and non-treated by RNA 5′-polyphosphatase, respectively. The two samples were normalized by amplification of 26S rRNA (26S) using outward-facing primers. The bands as indicated were sequenced by cloning into vectors. M: DNA molecular marker. (B) RT-qPCR analysis of the relative abundance of circularized primary transcripts after the treatment by 5′-polyphosphatase. n4: nad4-1, a8: atp8-1, cb: cob. +/-: nad4-1, atp8-1, or cob over 26S in the treated sample/nad4-1, atp8-1, or cob over 26S in the non-treated sample, respectively. The values are mean and SD of three biological replicates. (C) RNA gel blot assay of nad4, atp8, and cob transcripts. 2 µg mitochondrial RNAs were loaded in each lane. Bands corresponding to full-length mRNAs revealed by cRT-PCR are marked. n4, a8 and cb: RNA probes derived from coding regions of nad4, atp8, and cob genes, respectively. (D) Transcript termini deduced from cRT-PCR clones. Gene coding regions are shown as gray boxes, and 5′ and 3′ UTRs as bold lines. Positions of 5′ and 3′ ends relative to translation start codon AUG (+1) and stop codon UGA, UAA, or UAG (−1), and numbers of clones obtained at those positions are indicated. Positions of primers used for reverse transcription and PCR amplification are indicated by closed and open arrows, respectively. Positions of outward-facing primers used for RT-qPCR are indicated by open squares. Positions of RNA probes are as indicated. Scale bar = 100 nts.

Figure 2. Sequence analysis of the regions 20 nts upstream of transcription initiation sites. (A) Primary 5′ ends downstream of putative promoter core elements CRTA (R = A and G). Putative promoters are named as P-transcript name-distance between transcription initiating site and AUG (mature mRNAs) or the downstream exon (nad1T3), and P is short for Promoter; transcription initiating nucleotides are underlined, and putative promoter cores are written in bold; the number of molecules obtained at each initiation site together with the total number of clones sequenced for the recovered band are given. (B) MEME analysis discovered a 7-nt-long promoter motif upstream of 126 transcription initiation sites (for both mature and precursor RNAs). This motif is characterized by presence of a core element crTA (uppercase letters depict overrepresented nucleotide >1 bit).

Figure 3. Mapping of representative mature RNAs with processed 5′ ends. (A) Gel separation of cRT-PCR products. + and -: samples treated and non-treated by RNA 5′-polyphosphatase, respectively. The two samples were normalized by amplification of 26S rRNA (26S) using outward-facing primers. The bands as indicated were sequenced by cloning into vectors. n3: nad3, r12: rps12, 18S: 18S rRNA. M: DNA molecular marker. (B) RT-qPCR analysis of the relative levels of circularized processed transcripts after the treatment by 5′-polyphosphatase. +/-: nad3-rps12 or 18S over 26S in the treated sample/nad3-rps12 or 18S over 26S in the non-treated sample. The values are mean and SD of three biological replicates. (C) RNA gel blot assay of nad3-rps12 and 18S rRNA. 2 µg mitochondrial RNAs were loaded in each lane. Bands corresponding to mature RNAs revealed by cRT-PCR are marked. n3, r12 and 18S: RNA probes derived from coding regions of nad3, rps12 and rrn18 genes, respectively. (D) Transcript termini deduced from cRT-PCR clones. For nad3-rps12, the gene coding regions are shown as gray boxes, and 5′ and 3′ UTRs as bold lines; the mature 18S rRNA is shown as a black box. 5′ and 3′ ends of nad3-rps12 relative to nad3 AUG (+1) and rps12 UGA (−1) and the transcript termini of 18S rRNA in maize NB mitochondrial genome are indicated. Positions of primers used for reverse transcription and PCR amplification are indicated by closed and open arrows, respectively. Positions of outward-facing primers used for RT-qPCR are indicated by open squares. Positions of RNA probes are as indicated. Scale bar = 100 nts.

Figure 4. tRNAs and t-elements adjacent to the mapped transcript termini. Positions of transcript termini relative to the neighboring exons are indicated. tRNAs and t-elements are predicted by tRNAscan-SE and Mfold, respectively.

Table 2. Non-encoded nucleotides detected at 5ʹ→3ʹ junctions.

Steady-state transcripts	Non-encoded nucleotides *
nad3-rps12	A (3/19), AA (1/19), AAAA (1/19), AAAAA (1/19), AAAAAA (1/19), C (1/19), GCU (1/19), ACAAAC (1/19), GAUUUUUU (1/19), CCCUAAAAAAACA (1/19)
nad4-1	A (4/10)
nad4-2	AAA (1/16)
nad5-1	A (2/12), AAA (2/12)
nad5-2	A (2/15), AAA (3/15)
cox2-1	A (2/10)
cox2-2	A (1/10)
cox2-3	A (2/16)
cox2-4	A (4/27)
ccmFN-rps1-1	A (2/16), U (1/16)
ccmFN-rps1-2	A (1/11), GUCAC (1/11)
atp8-1	ACCCAA (2/29), C (1/29), AC (3/29), ACCC (1/29), ACCAA (2/29), ACC (2/29), ACCCUU (1/29), UAA (1/29)
atp8-2	AAAAAAAAA (2/24), ACCA (1/24), CCCA (1/24), ACCCAA (1/24), ACCAA (5/24), ACCC (2/24), ACCUAA (1/24), AC (1/24), ACCCU (1/24)
atp6-nad6-1	A (1/10)
mttB	A (9/23), AA (2/23), AAA (2/23), AAU (1/23), GAA (1/23), 1U (2/23)

Footnote: * Numbers in parentheses indicate the number of clones in which the non-encoded nucleotides are observed together with the total number of clones sequenced.

Figure 5. Mapping of nad5 precursors. (A) Gel separation of nad15T1, nad5T2, and nad5T3 cRT-PCR products. + and -: samples treated and non-treated by RNA 5′-polyphosphatase, respectively. The two samples were normalized by amplification of 26S rRNA (26S) using outward-facing primers. The bands as indicated were sequenced by cloning into vectors. M: DNA molecular marker. (B) Transcript termini of nad15T1, nad5T2, and nad5T3 deduced from cRT-PCR clones. Exons are shown as gray boxes, and 5′ and 3′ termini as bold lines. Positions of 5′ and 3′ ends in respect to the neighboring exons, and numbers of clones obtained at those positions are indicated. Positions of primers used for reverse transcription and PCR amplification are indicated by closed and open arrows, respectively. ex and int: exon and intron, respectively. Scale bar = 100 nts.

Figure 6. C→U RNA editing at nad2T2. (A) The six C→U editing sites identified at nad2 exon 5. Positions of the editing sites relative to nad2 UAA (−1) are indicated. All six sites are fully edited in the forty nad2 molecules analyzed. (B) Editing of the six C→U sites in nad1T4 precursor. Among the sixty-six nad2T2 molecules analyzed, twenty-nine were fully edited, four were not edited, and the other thirty-three were partially edited.