Table 1. Validation of several RNA targets of RNase III.
| 5′end | 3′end | Sta | Annotation/Alternative IDs | Adjacent genes/Orientation | Commentsb | Refc | Northern/mRNA stability exp | In vitro RNase III cleavage assay | Binding |
| ncRNAs | |||||||||
| 292655 | 292779 | − | RsaX28 | SA0240><>SA0241 | nd | ||||
| 413160 | 413255 | − | RsaL, SAU-5971 | rpsR><<SA0355 | Processed from large 3′UTR of SA0355 | (1) | Highly expressed | ||
| 436954 | 437055 | − | RsaM, Teg146, SAU63b | SA0377<<<SA0378 | Processed from large 3′UTR of SA0378 | (1,2) | Highly expressed | ||
| 501414 | 501696 | + | 4.5S | SA0434>>>SA0435 | 4.5S RNA | (2,3,4) | Highly expressed | N | Y |
| 511520 | 511626 | + | RsaX29, Teg43 | 5S rRNA>>>SA0439 | Sequence in part similar to 5S rRNA | (4) | |||
| 637038 | 637394 | + | RsaA | SA0543<><SA0544 | RsaA (short & large transcript) | (1,2,4) | Short and large transcripts less stable in wt than in Δrnc strains | Y, cleavages at the first hairpin | |
| 637016 | 637429 | − | asRsaA | SA0543<<<SA0544 | Start in SA0544 | (2) | nd | ||
| 765830 | 766139 | − | RsaN | SA0673><>SA0674 | Detected only in Δrnc strain | ||||
| 829498 | 829646 | − | asRsaH | SA0724<<>SA0725 | Less abundant than RsaH | nd | |||
| 829498 | 829643 | + | RsaH | SA0724<>>SA0725 | (1,2,4) | Detected in all strains | Y | ||
| 975383 | 975574 | + | RsaE | SA0859>><SA0860 | RsaE, RsaE-RsaF transcripts | (1,2,4) | Detected in all strains | RsaE-RsaF weak cleavages; one at the end of RsaE | |
| 1006734 | 1006851 | − | Teg102bis | SAS028><>SAS029 | Sequence partially repeated to the asSAS028 | (2,4) | Y | ||
| 1329666 | 1329870 | − | SA1167><>SA1168 | Overlapping Rho-independent terminator with SA1167 | nd | Y | |||
| 1437260 | 1437392 | + | RsaX31 | SA1265>>>SA1266 | Structured RNA with a UCCCA motif and a Rho-independent terminator. 10 repeats | (4) | nd | ||
| 1660498 | 1660771 | − | 6S | SA1455<<<aspS | SAS050 and 6S RNA (1660650–1660796) | (2,3,4) | Highly expressed | N (6S-SAS050) | Y |
| 2078734 | 2078872 | − | RNAIII | SA1841><>agrB | RNAIII (3′ domain) | (5) | Weakly expressed | RNAIII interacts with RNase III but is only cleaved when bound to mRNA targets (spa, rot) | Y |
| 2108482 | 2108586 | − | RsaX38 | ilvA><<5S rRNA | 5S rRNA like similar to RsaX29 | (4) | |||
| 2206379 | 2206710 | − | SprG3, Teg19b | SA1956><>SAS069 | Hypothetical sORF, antisense to SprF3, 5′ start at 2206385 | (3,4) | Highly expressed | SprFG3 hybrid completely degraded | |
| 2206379 | 2206710 | + | SprF3, Teg19a | SA1956>>>SAS069 | Antisense to SprG3, 5′ start at 2206708 | (2,3,4) | Highly expressed | ||
| 2370161 | 2370254 | − | RsaOas | SA2107><>SA2108 | nd | ||||
| 2370165 | 2370378 | + | RsaO | SA2107>>>SA2108 | 5′ start at 2370165 | Weakly expressed | |||
| 2421600 | 2421812 | + | RsaX41 | SA2155<><SA2156 | UCCCA motif | (4) | nd | ||
| rRNA and tRNA operons | |||||||||
| 549697 | 555585 | + | 5S_8tRNAs_16S_tRNA*_23S_5S | rRNA operon | Unprocessed in Δrnc strain | Y | |||
| 1152128 | 1152283 | − | tRNA | tRNA+terminator | N | Y | |||
| 1916277 | 1919063 | − | 26tRNAs_ 5S_23S_2tRNAs_16S | rRNA operon | Unprocessed in Δrnc strain | ||||
| 2109042 | 2114500 | − | 5S_23S_16S_2tRNAs | rRNA operon | Unprocessed in Δrnc strain | ||||
| 2230200 | 2230809 | − | 5tRNAs_5S_23S_16S | rRNA operon | Unprocessed in Δrnc strain | tRNATyr-tRNAGln cleaved but not at the expected site in vitro | Y | ||
| Riboswitches | |||||||||
| 15976 | 16095 | + | 5′UTR of SA0011 | SAS001>>>SA0011 | EL78, SAM riboswitch regulating the synthesis of homoserine-o-acetyl transferase | (2,4) | N* | Y | |
| 430802 | 430906 | + | 5′UTR of xrpT | SA0372<>>xprT | Purine riboswitch regulating the synthesis of xanthine phosphoribosyltransferase | (2,4) | N* | ||
| 1523781 | 1523956 | − | 5′UTR of SA1316 | SA1316<<<SA1317 | FMN riboswitch regulating the synthesis of SA1316 (hypothetical protein) | (2,4) | N* | Y* | |
| 1716104 | 1716314 | − | 5′UTR of thrS | thrS<<<dnaI | T-Box regulatory element regulating the synthesis of threonyl-tRNA synthetase | (2,4) | N | ||
| 2211998 | 2212074 | − | 5′ UTR glmS | glmS<<>SAS073 | GlcN6P riboswitch regulating the synthesis of glucosamine-6 phosphate; EL78; asRNA detected only in EP | (2,4) | mRNA highly expressed in SP but no difference between wt and Δrnc strains | ||
| mRNAs | |||||||||
| 122506 | 122736 | − | spa mRNA | SA0106><<spa | CoIP fragments along the mRNA | (6) | More expressed in Δrnc strain | Y | |
| 686734 | 686982 | + | 5′UTR tagG | tagH<>>tagG | Overlapping 5′UTR with tagH 5′UTR | Detected in Δrnc strain | tagG/tagH hybrid completely degraded | ||
| 686682 | 686968 | − | 5′UTR tagH | tagH<<>tagG | Overlapping 5′UTR with tagG 5′UTR | Detected in Δrnc strain | |||
| 716327 | 716597 | + | asSA0620 | SA0619>>>SA0621 | CoIP fragment covering the entire gene | nd | |||
| 716027 | 716571 | − | SA0620 | SsaA homologue; entire gene | nd | ||||
| 827582 | 827930 | + | clpP mRNA | Moderately expressed | N | ||||
| 828123 | 828309 | − | asclpP | clpP><<SA0724 | CoIP small fragments complementary to clpP mRNA | nd | |||
| 831523 | 839012 | + | operon gapR-gap-pgk-tpiA-pgm-eno | CoIP fragments covering all mRNAs within the operon | Enolase: highly expressed | ||||
| 1069600 | 1069996 | + | 5′UTR SA0943 | pdf1<>>SA0943 | Overlapping 5′UTR with pdf1 5UTR | 5′UTR only detected in Δrnc strain; mRNA detected in wt/rnc strains | |||
| 1069717 | 1069907 | − | 5′UTR-mRNA pdf1 | pdf1<<>SA0943 | Overlapping 5′UTR with SA0943 5′UTR; pdf1 mRNA accumulation in SP | pdf1 mRNA unprocessed in Δrnc strain | |||
| 1215989 | 1216195 | + | hmrB mRNA | CoIP fragment including the Rho-independant terminator | N | ||||
| 1216270 | 1216395 | + | rnc mRNA | CoIP fragment within the coding sequence of rnc mRNA, 5′ start at 1216196 | RNase III cleavage within the coding region | Y | Y | ||
| 1408765 | 1409144 | − | cspA mRNA | CoIP fragment covering the 5′UTR of cspA, 5′ start at 1409085 in wt strain | Unprocessed in Δrnc strain | Y | Y | ||
| 1409014 | 1409147 | + | ascspA | cspA<><SA1235 | EL78; more enriched in SP 5′ start at 1409015 | Very weakly expressed | Y; also cspA/as-cspA hybrid completely degraded | ||
| 1510679 | 1511027 | − | hu mRNA | 5′ start at 1511027 | Highly expressed, increased steady state level in Δrnc mutant | N | Y | ||
| 1510923 | 1511030 | + | ashu | hu<><gpsA | EL80; more enriched in SP | Weakly expressed | |||
| 2074084 | 2074375 | − | groES mRNA | EL80 | Accumulation of large transcript (operon) in EL78, EL79, EL80 | N | |||
| 2081487 | 2082375 | − | asagrA | EL80; CoIP fragment covers the whole mRNA | nd | ||||
| 2081528 | 2082221 | + | agrA mRNA | CoIP fragments along agrA mRNA | Upregulated in SP, no significant difference between wt and Δrnc strains | ||||
| 2298654 | 2298870 | − | secY mRNA | CoIP fragment within the coding region of secY mRNA | Highly expressed, more stable in Δrnc strain | Y | Y | ||
| 2298909 | 2298991 | + | as secY | EL80 | nd | ||||
| 2421872 | 2422221 | − | SA2156 | lctP homologue; entire gene covered | Y, weak cleavages; degradation? | ||||
| Hypothetical small ORFs | |||||||||
| 954606 | 954882 | − | SAS025 | SA0841><>SA0842 | nd | N | Y | ||
| 2000661 | 2000937 | − | SAS057 | SA1748<<>SA1749 | N | ||||
| 1006325 | 1006481 | + | SAS028 | SA0885>>>SAS029 | SAS028: hypothetical protein similar to lactococcin 972 | (1,2) | Highly expressed, steady state level higher in Δrnc strain than in the wt strain | Y; weak cleavages; degradation? | |
| 1006234 | 1006479 | − | asSAS028/SAU-02/teg102 | SA0885><>SAS029 | Partially repeated with Teg102bis RNA, 5′ start at 1409015 | (1,2) | Less abundant than SAS028 mRNA | ||
The annotation of the genome is taken from strain N315.
(a)
Strand (+/−) of the genome;
(b)
Experiments were performed in RN6390 strain and the mutant Δrnc strain transformed with a plasmid expressing the wt enzyme (wt, strain EL79), the mutant D63A (strain EL80), or E135A enzyme (strain EL78); nd is for not detected.
(c)
References: (1) Abu-Qatouseh et al. [36]; (2) Beaume et al. [37]; (3) Pichon and Felden [42]; (4) Geissmann et al. [43]; (5) Novick et al. [44]; (6) Huntzinger et al. [23]. N: No cleavage or binding detected; Y: cleavage (or binding) detected.
*: Assays were done in the presence or absence of the respective ligand. RNase III cleavage and binding assays were done as described in Materials and Methods.